Enzymatic conjugation of polypeptides

ABSTRACT

The present application relates to methods for the functionalization of immunoglobulins, in particular with drugs. Also disclosed herein are linking reagents, functionalized antibodies, pharmaceutical compositions, and method of treating disease and/or conditions

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Nos.61/671,122, filed Jul. 13, 2012; 61/661,569, filed Jun. 19, 2012; and61/579,908, filed Dec. 23, 2011; all of which are incorporated herein byreference in their entirety; including any drawings.

REFERENCE TO SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledINNAT010A.TXT, created Dec. 20, 2012, which is 20 KB in size. Theinformation in the electronic format of the Sequence Listing isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present application relates to the field of chemistry, biochemistry,and medicine. Disclosed herein are methods for the functionalization ofimmunoglobulins, in particular with drugs. Also disclosed herein arelinking reagents, functionalized antibodies, pharmaceuticalcompositions, and method of treating disease and/or conditions.

BACKGROUND

Immunoglobulins conjugated to a drug of interest, generally known asantibody drug conjugates (ADCs), are a promising area of therapeuticresearch. Recent developments in ADC technology have focused on linkertechnology that provides for intracellular cleavage or more recently,non-cleavable linkers that provide greater in vivo stability and reducedtoxicity. The feasibility of a non-cleavable linker-based approach,however, may be more dependent on the cellular target than in the caseof cleavable linkers. ADCs with noncleavable linkers must beinternalized and degraded within the cell, whereas compounds withcleavable linkers may be active against targets that are poorlyinternalized through extracellular drug release and drug entry intotumor cells. Similarly, killing of bystander antigen-negative cellsthrough targeting of antigen-positive cells (collateral toxicity) ispresumably only possible with cleavable linkers. As a consequence, it isgenerally believed that no general linker design exists for all ADCs andthat each antibody must be examined separately. Additionally, theefficacy of a drug linked to a toxin may vary, e.g. depending on thecell type or particular tumor cell, such that it may also be necessaryto test a variety of drugs against a given target and further incombination with a particular linker system. Development of ADCstherefore remains an expensive and time-consuming process and there is aneed in the field for improved linker systems.

Transglutaminases (TGases) have been exploited for some time in the foodindustry for their ability to cross-link proteins. Such utilization hasavoided the need to cross-link in quantitative or stoichiometricfashion. TGases have been shown to be capable of conjugating glutamineand lysine residues, including on antibodies (see, e.g., Josten et al.(2000) J. Immunol. Methods 240, 47-54; Mindt et al (2008) Bioconjug.Chem. 19, 271-278; Jeger et al (2010) Angew. Chem. Int. Ed. 49:9995-9997); Kamiya et al (2003) Enzyme. Microb. Technol. 33, 492-496 andUS patent publication no. 2011/0184147. While previous attempts tocross-link proteins have studied protein motifs that gave rise toconjugation and identified peptides that can be conjugated, the ruleswhich govern selection by TGases of glutamine residues for modificationare still largely unknown. Additionally, little is known about TGases'ability to take up different substrates, or their effect on the abilityto TGases to conjugate in quantitative fashion.

SUMMARY OF THE INVENTION

The present invention provides methods using TGase to stoichiometricallyfunctionalize acceptor glutamines on antibodies with difficultsubstrates. The invention arises from the finding that the environmentof acceptor glutamines within antibodies and the nature of thelysine-derivative donor substrates both independently and in combinationinfluence the efficacy of transglutaminase (TGase)-mediated conjugation.

It has been discovered that conjugation of moieties (e.g. chemicalentities) to antibodies using TGase in combination withlysine-derivative linkers provides at best only partial conjugation toacceptor glutamines within antibodies. The conjugation appears to bedependent, among other things, on the nature of the substrate: whilesmaller, uncharged and non-hydrophobic substrates such as biotin can bepartially coupled using known methods, some substrates such as chargedsubstrates are not coupled at all. Hydrophobic and/or larger substratesappear to be poorly coupled leading to heterogeneous mixtures ofantibodies. Coupling reaction parameters were optimized but could notresolve the problems of low levels of coupling and thus producthomogeneity. Large, rigid and/or hydrophobic molecules, particularlythose also containing polycycles or macrocycles, (along the lines ofcommon cytotoxic drugs) could not be coupled with a high level ofcompletion.

Linkers for improving the direct coupling of such molecules weredeveloped. The linkers that yielded improved coupling provided a spacerand/or lacked polar and/or polycycles or macrocycles (or generallygroups conferring structural rigidity) close to the site of BTGinteraction, that is, proximal to the primary amine, and/or providedspacers that distanced the site of BTG interaction from substituentsthat inhibit BTG-mediated coupling. Such linkers enabled improvedcoupling of smaller compounds with charges and macrocycles (up to 70%conjugation) onto deglycosylated antibodies. The linker also enabled thedesign of reactive linkers that enabled a multi-step approach thatpermitted complete and stoichiometric conjugation of larger molecules.

Examples include linkers of Formulae I, II, III or IV, optionallywherein any of (C)_(n), L, V or Y (and any combinations thereof) mayfunction as the spacer. In one embodiment, a (C)_(n), and/or L groupcomprise a linear hydrocarbon chain; in one embodiment, a (C)_(n) and/orL group comprise a plurality of (CH₂—CH₂—O—) groups, optionally(CH₂—CH₂—O—)_(n) group wherein n is an integer selected among the rangeof 1 to 24; in one embodiment, a (C)_(n) and/or L group comprise anamino acid residue (e.g. a lysine residue) or a di-, tri-, tetra, oroligopeptide. In one embodiment, a V group comprises a di-, tri-, tetra,or oligopeptide, optionally wherein the peptide is cleavable in a cell.In one embodiment, (C)_(n) comprises a lysine residue or derivative andV comprises a di-, tri-, tetra, or oligopeptide, optionally wherein L ispresent or absent. Optionally, the di-, tri-, tetra, or oligopeptide(s)comprise or consist of amino acid residues with non-negatively chargedside chains (amino acids other than aspartic acid or glutamic acid).Optionally, the di-, tri-, tetra, or oligopeptide(s) comprise or consistof amino acid residues selected from amino acid residues with positivelycharged side chains, amino acid residues with polar uncharged sidechains, and amino acid residues with hydrophobic side chains.

It was further discovered that substantially complete coupling of largerand hydrophobic molecules with cyclic groups (e.g. auristatin toxin)could be achieved when modified Fc domains were used that avoided anegatively charged aspartic acid group at the +2 position relative tothe acceptor glutamine. The Fc domain modifications were designed toabolish heavy chain N297-linked glycosylation, such that no enzymatic(PNGase F) deglycosylation was needed. PNGase F deglycosylation modifiesthe side chain of the asparagine at position 297 (EU index) whichbecomes a negatively charged aspartic acid residue. Use of a N297Smutant with one acceptor glutamine per heavy chain (at residue 295 (EUindex)) yielded an antibody composition in which more than than 90% ofantibodies had one functionalized acceptor glutamine on each heavychain.

Furthermore, it was observed that both the environment of the linker andthe environment of the acceptor glutamine contribute to conjugation.Combining the modified Fc domains with linker structure providedadditive improvement in coupling in terms of completion of coupling ofall available acceptor glutamines on antibodies within a composition fora linker comprising a negatively charged moiety-of-interest.

In addition to improved linkers and antibodies for direct (one-step)TGase-mediated conjugation, a strategy was developed based on amulti-step approach. Improved linkers were developed that lacked polarand/or polycycles or macrocycles (rigid groups) close to the site of BTGinteraction, followed by reaction with a reaction partner that containsthe moiety of interest (see e.g., FIG. 10B for an example of amulti-step approach). Linkers were also developed that included a spacerand could be used for conjugation of smaller linkers that comprisedcyclic groups (e.g. cycloalkynes). Different linear linkers were used todemonstrate the concept. This multi-step approach permittedsignificantly higher completion of coupling in a composition ofantibodies. In one advantageous configuration, a polycyclic reactivegroup was placed on a longer linker comprising a spacer and acomplementary reactive group (non-polycyclic) was placed on the reactionpartner. In one advantageous configuration, a polycyclic reactive groupwas placed on the reaction partner and non-polycyclic reactive group wasplaced onto the linker. The multi-step approach permitted the completionof coupling of larger and/or hydrophobic moieties onto two acceptorglutamines per antibody chain (four acceptor glutamines).

The multi-step approach displays advantages that permitted preparationof compounds that have Z moieties that are subject to degradation whenmaintained at 37° C., the temperature at which TGase reaction was mostactive. Examples of such compounds include toxins, e.g. auristatins. Themulti-step approach also permitted preparation of conjugates using lowerequivalents of (Z) moieties were used (compared to antibodies). Whiledirect coupling required 80 equivalents of TGase substrate toPNGaseF-deglycosylated wild-type antibody for significant coupling (tothe extent that this was even possible), the multi-step approachdecreased the number of equivalents of Z moieties needed. For compoundsuch as hydrophobic compounds, generally large organic molecules thattypify toxins, high concentrations can be problematic and moreover oftenrequire organic solvents, which solvents in turn inhibit TGase activity.

In one aspect, present invention provides a site-specific labeling andfunctionalization approach that is particularly useful forfunctionalizing immunoglobulins with drugs, particularly peptides andpolypeptides, relatively large chemical entities, negatively chargedchemical entities, chemical entities comprising macrocycles or one or aplurality of cyclic groups and/or hydrophobic chemical entities, e.g.typical cytotoxic drugs such as duocarmycins, maytansanoids, alkylatingagents, taxanes, auristatins (e.g., MMAE, MMAF) and the like (e.g.analogues thereof) that are derived from natural sources, or analoguesor derivatives thereof.

The present invention relates in one embodiment to a method forconjugating a moiety of interest (Z) to an antibody, comprising thesteps of:

a) producing (e.g. in a recombinant host cell) an antibody comprising anacceptor glutamine residue (e.g. within the primary sequence of theantibody) flanked at the +2 position by a non-glycosylated, non-asparticacid, amino acid residue; and

b) reacting said antibody comprising an acceptor glutamine residue (e.g.within the primary sequence of the antibody) flanked at the +2 positionby a non-glycosylated, non-aspartic acid, amino acid residue with alinking reagent comprising a primary amine, in the presence of atransglutaminase enzyme capable of causing the formation of a covalentbond between the acceptor glutamine residue and the linking reagent (atthe primary amine of the linking reagent), under conditions sufficientto obtain an antibody comprising an acceptor glutamine residue linked(covalently) to the linking reagent. In one embodiment, the linkingreagent comprises a moiety-of-interest (Z), wherein Z is a hydrophobicor charged organic compound, and/or is an organic compound having amolecular weight of at least 400, 500, 700 or 800 g/mol. In oneembodiment, the linking reagent comprises a protected or unprotectedreactive group (R). Optionally, in step (b), the linking reagent,optionally the linking reagent comprising a moiety of interest (Z), isprovided in an amount which is less than 80, 40, 20, 10, 5, 4, or 3molar equivalents to the antibody. In one embodiment, the antibody hastwo acceptor glutamines and the linking reagent comprising a moiety ofinterest (Z) is provided in an amount which is less than 40 equivalentsto the antibody, optionally between 20 and 40 or between 20 and 75equivalents to the antibody. In one embodiment, the linking reagentcomprises a reactive group (R). Optionally, (e.g. in a multi-step methodof the invention), the linking reagent comprising a moiety of interest(R) is provided in an amount which is between 2 and 40 or between 2 and20 molar equivalents to the antibody, optionally wherein the antibodycomprises two acceptor glutamines. Optionally, the linking reagentcomprising a moiety of interest (R) is provided in an amount which isbetween 4 and 40 or between 4 and 20 molar equivalents to the antibody,optionally wherein the antibody comprises four acceptor glutamines.

Accordingly, the invention also provides an antibody or antibodyfragment comprising an acceptor glutamine residue flanked at the +2position by a non-aspartic acid residue, wherein the acceptor glutamineresidue is functionalized, optionally via a linking reagent, with acompound comprising a moiety-of-interest. Optionally, the moiety ofinterest is a peptide, a polypeptide, an organic compound, amoiety-of-interest that improves the pharmacokinetic properties, atherapeutic moiety or a diagnostic moiety. Optionally, themoiety-of-interest is a hydrophobic or charged organic compound, and/oris an organic compound having a molecular weight of at least 400, 500,700 or 800 g/mol. Optionally, the residue at the +2 position is anon-aspartic acid residue. In one embodiment, the residue at the +2position is a non-aspartic acid, non-glutamine residue. In oneembodiment, the residue at the +2 position is a non-aspartic acid,non-asparagine residue. In one embodiment, the residue at the +2position is a non-negatively charged amino acid (an amino acid otherthan an aspartic acid or a glutamic acid). Optionally, the acceptorglutamine is in an Fc domain of an antibody heavy chain, optionallyfurther-within the CH2 domain. Optionally, the antibody is free of heavychain N297-linked glycosylation. Optionally, the acceptor glutamine isat position 295 and the residue at the +2 position is the residue atposition 297 (EU index numbering) of an antibody heavy chain.Optionally, the acceptor glutamine is at position 297 and the residue atthe +2 position is the residue at position 299 (EU index numbering) ofan antibody heavy chain. Optionally, said moiety-of-interest iscovalently bound to the acceptor glutamine residue via a linkercomprising a NH—(C)_(n) group, wherein (C)_(n) is a substituted orunsubstituted carbon chain, wherein any carbon of the chain isoptionally substituted with an alkoxy, hydroxyl, alkylcarbonyloxy,alkyl-S—, thiol, alkyl-C(O)S—, amine, alkylamine, amide, or alkylamide;and n is an integer from among the range of 2 to 200, optionally 2 to100, optionally 2 to 50, optionally 2 to 20.

The present invention relates in one embodiment to a method forconjugating a moiety of interest (Z) to an antibody, comprising thesteps of:

a) providing an antibody having (e.g. within the primary sequence of aconstant region) at least one acceptor amino acid residue (e.g. anaturally occurring amino acid) that is reactive with a linking reagent(linker) in the presence of a coupling enzyme, e.g., a transamidase; and

b) reacting said antibody with a linking reagent (e.g. a linkercomprising a primary amine) comprising a reactive group (R), optionallya protected reactive group or optionally an unprotected reactive group,in the presence of an enzyme capable of causing the formation of acovalent bond between the acceptor amino acid residue and the linkingreagent (other than at the R moiety), under conditions sufficient toobtain an antibody comprising an acceptor amino acid residue linked(covalently) to a reactive group (R) via the linking reagent.Optionally, said acceptor residue of the antibody or antibody fragmentis flanked at the +2 position by a non-aspartic acid residue.Optionally, the residue at the +2 position is a non-aspartic acidresidue. In one embodiment, the residue at the +2 position is anon-aspartic acid, non-glutamine residue. In one embodiment, the residueat the +2 position is a non-aspartic acid, non-asparagine residue. Inone embodiment, the residue at the +2 position is a non-negativelycharged amino acid (an amino acid other than an aspartic acid or aglutamic acid). Optionally, the acceptor glutamine is in an Fc domain ofan antibody heavy chain, optionally further-within the CH2 domainOptionally, the antibody is free of heavy chain N297-linkedglycosylation. Optionally, the acceptor glutamine is at position 295 andthe residue at the +2 position is the residue at position 297 (EU indexnumbering) of an antibody heavy chain.

In one aspect, present invention relates in one embodiment to a methodfor conjugating a moiety of interest (Z) to an antibody, comprising thesteps of:

a) providing an antibody having at least one acceptor glutamine residue;and

b) reacting said antibody with a linker comprising a primary amine (alysine-based linker) comprising a reactive group (R), preferably aprotected reactive group, in the presence of a TGase, under conditionssufficient to obtain an antibody comprising an acceptor glutamine linked(covalently) to a reactive group (R) via said linker Optionally, saidacceptor glutamine residue of the antibody or antibody fragment isflanked at the +2 position by a non-aspartic acid residue. Optionally,the residue at the +2 position is a non-aspartic acid residue. In oneembodiment, the residue at the +2 position is a non-aspartic acid,non-glutamine residue. In one embodiment, the residue at the +2 positionis a non-aspartic acid, non-asparagine residue. In one embodiment, theresidue at the +2 position is a non-negatively charged amino acid (anamino acid other than an aspartic acid or a glutamic acid). Optionally,the acceptor glutamine is in an Fc domain of an antibody heavy chain,optionally further-within the CH2 domain Optionally, the antibody isfree of heavy chain N297-linked glycosylation. Optionally, the acceptorglutamine is at position 295 and the residue at the +2 position is theresidue at position 297 (EU index numbering) of an antibody heavy chain.

The antibody comprising an acceptor residue or acceptor glutamineresidue linked to a reactive group (R) via a linker comprising a primaryamine (a lysine-based linker) can thereafter be reacted with a reactionpartner comprising a moiety of interest (Z) to generate an antibodycomprising an acceptor residue or acceptor glutamine residue linked to amoiety of interest (Z) via the linker. Thus, in one embodiment, themethod further comprises a step (c): reacting (i) an antibody of step b)comprising an acceptor glutamine linked to a reactive group (R) via alinker comprising a primary amine (a lysine-based linker), optionallyimmobilized on a solid support, with (ii) a compound comprising a moietyof interest (Z) and a reactive group (R′) capable of reacting withreactive group R, under conditions sufficient to obtain an antibodycomprising an acceptor glutamine linked to a moiety of interest (Z) viaa linker comprising a primary amine (a lysine-based linker). Preferably,said compound comprising a moiety of interest (Z) and a reactive group(R′) capable of reacting with reactive group R is provided at a lessthan 80 times, 40 times, 20 times, 10 times, 5 times or 4 molarequivalents to the antibody. In one embodiment, the antibody comprisestwo acceptor glutamines and the compound comprising a moiety of interest(Z) and a reactive group (R′) is provided at 10 or less molarequivalents to the antibody. In one embodiment, the antibody comprisestwo acceptor glutamines and the compound comprising a moiety of interest(Z) and a reactive group (R′) is provided at 5 or less molar equivalentsto the antibody. In one embodiment, the antibody comprises four acceptorglutamines and the compound comprising a moiety of interest (Z) and areactive group (R′) is provided at 20 or less molar equivalents to theantibody. In one embodiment, the antibody comprises four acceptorglutamines and the compound comprising a moiety of interest (Z) and areactive group (R′) is provided at 10 or less molar equivalents to theantibody. In one embodiment, steps (b) and/or (c) are carried out inaqueous conditions. Optionally, step (c) comprises: immobilizing asample of an antibody comprising a functionalized acceptor glutamineresidue of Formula II on a solid support to provide a sample comprisingimmobilized antibodies, reacting the sample comprising immobilizedantibodies with a compound of Formula III, optionally recovering anyunreacted compound and re-introducing such recovered compound to thesolid support for reaction with immobilized antibodies, and eluting theantibody conjugates to provide an antibody composition of Formula IVbcomprising a Z moiety.

The invention provides, inter alia, the compositions having narrowdistributions of numbers of conjugates per antibody that result from themethods of the invention for conjugating a moiety of interest (Z) to anantibody. Such compositions are advantageous for human therapy. Inparticular, in one aspect the invention provides antibody compositions(e.g. compositions of a plurality of tetrameric, full-length antibodies)having a well-defined distribution of number of conjugates per antibody,and in particular, a narrow Drug-Antibody Ratio (DAR) distribution. Inparticular, the method permits substantially complete conjugation ofantibodies. In one aspect the invention provides a composition wherein ahigh portion of antibodies in the composition (e.g. at least 80%, 85%,90%, 95% of the antibodies) comprise at least one moiety of interestconjugated, via a linker (e.g. a linker of Formula Ia, Ib or Ic), to oneor two acceptor glutamines on each heavy chain, wherein the compositionis substantially free of antibodies comprising a number of moieties ofinterest that is greater than 2 times, optionally 1.5 times, the meannumber of conjugates per antibody (e.g., the mean DAR). The inventionprovides a composition wherein a high portion of antibodies in thecomposition (e.g. at least 80%, 85%, 90%, 95% of the antibodies)comprise at least one moiety of interest conjugated, via a linker, to anacceptor glutamine within a heavy chain, wherein compositions of theinvention are preferably also free of antibodies having conjugated lightchains. For example, the invention provides a composition of tetramericantibodies covalently linked to a moiety of interest (Z), wherein thecomposition is characterized by a mean DAR of close to 2 (e.g., between1.5 and 2.0, or between 1.7 and 2.0, between 1.8 and 2.0, or between 1.9and 2.0) wherein the composition is substantially free of antibodieshaving more than 2 moieties of interest per antibody. In anotherexample, the invention provides a composition of tetrameric antibodiescovalently linked to a moiety of interest (Z), wherein the compositionis characterized by a mean DAR of close to 4 (e.g., between 3.0 and 4.0,or between 3.4 and 4.0, or between 3.6 and 4.0) wherein the compositionis substantially free of antibodies having more than 4 moieties ofinterest per antibody. Optionally, said acceptor glutamine residue(s) ofthe antibodies or antibody fragments in the composition is flanked atthe +2 position by a non-aspartic acid residue. Optionally, the residueat the +2 position is a non-aspartic acid residue. In one embodiment,the residue at the +2 position is a non-aspartic acid, non-glutamineresidue. In one embodiment, the residue at the +2 position is anon-aspartic acid, non-asparagine residue. In one embodiment, theresidue at the +2 position is a non-negatively charged amino acid (anamino acid other than an aspartic acid or a glutamic acid). Optionally,the acceptor glutamine is in an Fc domain of an antibody heavy chain,optionally further-within the CH2 domain. Optionally, the antibody isfree of heavy chain N297-linked glycosylation. Optionally, the acceptorglutamine is at position 295 and the residue at the +2 position is theresidue at position 297 (EU index numbering) of an antibody heavy chain.

The methods of the invention also provide a way to rapidly screen arange of drugs, spacers and/or linkers given a particular startingantibody. Such comparisons are made possible because the presentapproach provides for homogenous antibody: drug stoichiometry, havingnot only advantages in production processes but also allowing the directcomparison of biological (e.g. cytotoxic) activity between an antibodyfunctionalized with different linker and/or drug combinations.

The technique further provides improved production processes, includingeconomic benefit in that lower quantities of substrate (e.g., drugs orother moieties to be conjugated) can be used, compared to currentlyavailable methods. Notably it will be possible to use as little as 20,10, 5, 4, 3 or 2 equivalents of a moiety of interest:antibody, therebyproviding savings for expensive reagents such as drugs.

Decreasing the quantity of substrate is also valuable in that it permitslower concentrations of drugs to be used, which for hydrophobic orotherwise poorly water-soluble drugs in turn permits lowerconcentrations of solvents to be used. The technique thus furtherprovides processes for functionalization of antibodies with moieties inaqueous conditions on in conditions of low organic solvent concentration(or substantially free of organic solvent). Because certain drugs (e.g.hydrophobic drugs) that require organic solvents for solubility athigher concentration can be coupled using the present inventionsubstantially in the absence of organic solvent, in any of theembodiments herein the invention provides antibody compositions (forexample manufacturing or biological intermediates) functionalized withdrugs (e.g. hydrophobic drugs) that are substantially free of organicsolvent and/or are in aqueous buffer (e.g. containing 20%, 10%, 5% orless organic solvent, e.g. DMSO). Presence of organic solvents isundesirable in manufacturing and at higher concentrations may alsoinhibit the activity of TGase. In one embodiment, any of the method ofthe inventions are performed in aqueous buffer (e.g. substantially freeof organic solvent, for example containing 20%, 10%, 5% or less solvent,for example containing between 0.01 and 10% organic solvent, for examplebetween 0.01 and 10% DMSO). In one embodiment, any of the method of theinventions, a TGase is provided at a concentration of at least 2 U/ml, 4U/ml, or at least 6 U/ml, or optionally between 2, 4, 5 or 6 U/ml and100 U/ml, or optionally between 2, 4, 5 or 6 U/ml and 20 U/ml, oroptionally between 2, 4, 5 or 6 U/ml and 12 U/ml, or optionally between2, 4, 5 or 6 U/ml and 10 U/ml, or optionally between 2 or 4 U/ml and 6U/ml. In one embodiment, any of the method of the inventions, aTGase-mediated reaction is carried out at neutral pH (about pH 7.4). Inone embodiment, any of the method of the inventions, a TGase-mediatedreaction is carried out at about 37° C.

The technique further provides improved production processes forachieving complete functionalization with large, charged or hydrophobicmoieties of interest. In one embodiment, any of the method of theinventions, a TGase-mediated reaction (e.g., a TGase reaction step of amethod described herein) is carried out for less than 48 hours,optionally less than 24 hours, optionally between 2 and 18 hours,between 2 and 24 hours, between 2 and 18 hours or between 4 and 18hours, optionally at about 37° C.

Certain aspects of the invention are directed to a linking reagent thatcan be attached, by the action of a TGase, to a polypeptide at aglutamine residue (Q) within the sequence of the antibody (Ab). Thelinking reagent comprises a lysine derivative (Lys) or a functionalequivalent thereof, that is connected to at least one reactive group (R)or a moiety-of-interest (Z). The lysine derivative (Lys) or a functionalequivalent can comprise generally any primary amine chain which is asubstrate for TGase, e.g. comprising an alkylamine, oxoamine. In oneembodiment, a plurality of reactive groups, preferably non-complementaryreactive groups, can be attached to the linking reagent. The reactivegroup is preferably a functionality that is insensitive to water butselectively undergoes a very high conversion addition reaction with acomplementary reagent. The functional equivalent of a lysine derivativemay comprise a 2 to 20 carbon chain, or a functional equivalent thereof,with an H₂N or H₂NCH₂ (aminomethylene)) group, or a protected H₂N orH₂NCH₂ group that can be derived from the H₂N or aminomethylenepositioned at one or more ends of the carbon chain. The functionalequivalent of the carbon chain may comprise a chain of 3 to 20 atomswhere one or more of the atoms other than the primary amine can be otherthan carbon, for example oxygen, sulfur, nitrogen, or other atoms, e.g.with an H₂NOCH₂ group, or a protected H₂NOCH₂ group positioned at one ormore ends of the carbon chain. The oxygen, sulfur, or nitrogen atom canbe of an ether, ester, thioether, thioester, amino, alkylamino, amido oralkylamido functionality within the carbon chain.

One exemplary functional equivalent of the carbon chain is an oligo(ethylene oxide) chain The functionality within the carbon chain can beincluded to couple the reactive group to the H₂N H₂NOCH₂ or H₂NCH₂ groupor protected H₂N, H₂NOCH₂ or H₂NCH₂ group. The carbon chain, or itsfunctional equivalent, can be substituted or unsubstituted. Thesubstituents can be alkyl groups, aryl groups, alkyl aryl groups,carboxylic acid groups, amide groups, hydroxy groups, or any othergroups that do not compete with the amino group for, or inhibit,conjugation with a glutamine residue of the protein. Typically, when asubstituent is present, its presence is in a convenient startingmaterial, such as the carboxylic acid group of lysine, from which thelysine derivative results. The amine at the end of a carbon chain orfunctional equivalent is necessarily included in the linking reagent.

Examples of starting materials for the functional equivalent of lysinecan be an α,ω-diaminoalkane, for example, 1,2-diaminoethane,1,3-diaminopropane, 1,4-diaminobutane, 1,5-diaminopentane,1,6-diaminohexane, 1,7-diaminoheptane, 1,8-diaminooctane,1,9-diaminononane, 1,10-diaminodecane, 1,11-diaminoundecane, or1,12-diaminododecane. Other starting materials for the functionalequivalent of a lysine derivative can be α,ω-diamino oligo (ethyleneoxide), for example, H₂N(CH₂CH₂O)_(x)CH₂CH₂NH₂ where x is an integerselected among the range of 1 to 6. The α,ω-diamino oligo (ethyleneoxide) can be a single oligomer or it can be a mixture of oligomerswhere x defines an average size. An exemplary protected H₂NCH₂ is thetert-butylcarbamate protected amine of tert-butylN-(5-aminopentyl)carbamate (N-Boc-cadaverin).

Linking reagents used for direct (one-step) linking of a moiety ofinterest (Z) to an antibody will advantageously comprise an element thatfunctions as a spacer to distance a large, charged or hydrophobicorganic moiety-of-interest (Z) from the acceptor glutamine. The spacermay be embodied in the lysine derivative or functional equivalentthereof, or in a further element of the linker (e.g. an L, V and/or Ygroup). In one embodiment, the element that functions as a spacer is alysine derivative (Lys) or a functional equivalent thereof having astructure NH—(C)_(n)—, wherein (C)_(n) is a substituted or unsubstitutedalkyl or heteroalkyl chain, wherein any carbon of the chain isoptionally substituted with an alkoxy, hydroxyl, alkylcarbonyloxy,alkyl-S—, thiol, alkyl-C(O)S—, amine, alkylamine, amide, or alkylamide,and where n is an integer greater than 10, optionally an integer fromamong the range of 10 to 20. In one embodiment, the linking reagentcomprises an L, V and/or Y group that functions as a spacer and ispositioned between the NH—(C)_(n)— group and the moiety-of-interest (Z),wherein L is a carbon-comprising framework of 1 to 200 atoms substitutedat one or more atoms, optionally wherein the carbon comprising frameworkis a linear hydrocarbon, a symmetrically or asymmetrically branchedhydrocarbon, monosaccharide, a glycan, disaccharide, linear or branchedoligosaccharide (asymmetrically branched or symmetrically branched),other natural linear or branched oligomers (asymmetrically branched orsymmetrically branched), amino acid residue, di-, tri- or oligopeptide,or any dimer, trimer, or higher oligomer (linear, asymmetricallybranched or symmetrically branched) for example resulting from anychain-growth or step-growth polymerization process; V is a non-cleavablemoiety or a conditionally-cleavable moiety, optionally following priorconditional transformation, which can be cleaved or transformed by achemical, photochemical, physical, biological, or enzymatic process(e.g. cleavage of V ultimately leading to release of one or moremoieties subsequently or ultimately linked to V, for example a Zmoiety). In some embodiments, V is, preferably, a di-, tri-, tetra-, oroligopeptide as described below in the section entitled “The V Moiety”;and Y is a spacer system (e.g., a self-eliminating spacer system or anon-self-elimination spacer system) which is comprised of 1 or morespacers.

In one embodiment, the invention provides a linking reagent, or aprotein-conjugated linking reagent, having the general Formula Ia:

G-NH—(C)_(n)—X-L-(V—(Y—(Z)_(z))_(q))_(r)  Formula Ia

or a pharmaceutically acceptable salt or solvate thereof;

wherein:

G is an H, amine protecting group, an antibody or antibody fragmentattached via an amide bond (e.g. through an acceptor glutamine residuein the primary sequence of the antibody or antibody fragment);

(C)_(n) is a substituted or unsubstituted alkyl or heteroalkyl chain,optionally wherein any carbon of the chain is substituted with analkoxy, hydroxyl, alkylcarbonyloxy, alkyl-S—, thiol, alkyl-C(O)S—,amine, alkylamine, amide, or alkylamide;

n is an integer from among the range of 2 to 20;

X is NH, O, S, or absent, or a bond;

L is independently absent, a bond or a continuation of a bond, or acarbon comprising framework of 5 to 200 atoms substituted at one or moreatoms;

r is an integer selected from among 1, 2, 3 or 4;

q is an integer selected from among 1, 2, 3 or 4;

z is an integer selected from among 1, 2, 3 or 4; and

V is independently absent, a bond or a continuation of a bond, anon-cleavable moiety or a conditionally-cleavable moiety;

Y is independently absent, a bond or a continuation of a bond, or aspacer system which is comprised of 1 or more spacers; and

Z is a moiety that improves the pharmacokinetic properties, atherapeutic moiety or a diagnostic moiety, optionally wherein Z is anorganic compound that is electrically negatively charged, hydrophobicand/or that has a molecular weight of at least 400 g/mol.

In one embodiment, n is an integer from among the range of 10 to 20. Inone embodiment, (C)_(n) is a heteroalkyl chain that comprises a(CH₂—CH₂—O—), group, wherein x is an integer from among the range of 1to 6. In one embodiment, at least one of L, V or Y are present. In oneembodiment, n is an integer from among the range of 2 to 6 and at leastone of L, V or Y are present.

In one embodiment, the invention provides an antibody or antibodyfragment comprising a functionalized acceptor glutamine residue, thefunctionalized acceptor glutamine residue having Formula IVa,

(Q)-NH—(C)_(n)—X-L-(V—(Y—(Z)_(z))_(q))_(r)  Formula IVa

or a pharmaceutically acceptable salt or solvate thereof,

wherein:

Q is glutamine residue present in an antibody or antibody fragment;

(C)_(n) is a substituted or unsubstituted alkyl or heteroalkyl chain,optionally wherein any carbon of the chain is substituted with analkoxy, hydroxyl, alkylcarbonyloxy, alkyl-S—, thiol, alkyl-C(O)S—,amine, alkylamine, amide, or alkylamide;

n is an integer from among the range of 2 to 20;

X is NH, O, S, absent, or a bond;

L is independently absent, a bond or a continuation of a bond if X is abond, or a carbon comprising framework of 5 to 200 atoms substituted atone or more atoms;

r is an integer selected from among 1, 2, 3 or 4;

q is an integer selected from among 1, 2, 3 or 4;

z is an integer selected from among 1, 2, 3 or 4; and

V is independently absent, a bond or a continuation of a bond, anon-cleavable moiety or a conditionally-cleavable moiety;

Y is independently absent, a bond or a continuation of a bond, or aspacer system which is comprised of 1 or more spacers; and

Z is a moiety that improves the pharmacokinetic properties, atherapeutic moiety or a diagnostic moiety, optionally, wherein Z is anorganic compound that is electrically negatively charged, hydrophobicand/or that has a molecular weight of at least 400 g/mol.

In one embodiment, n is an integer from among the range of 10 to 20. Inone embodiment, (C)_(n) is a heteroalkyl chain that comprises a(CH₂—CH₂—O—)_(x) group, wherein x is an integer from among the range of1 to 6. In one embodiment, at least one of L, V or Y are present. In oneembodiment, n is an integer from among the range of 2 to 6 (i.e. 2, 3,4, 5 or 6) and at least one of L, V or Y are present.

Optionally, in any of the linking reagents, protein-conjugated linkingreagents, antibodies or antibody fragments of the invention, L comprisesa linear carbon comprising framework of 5 to 30 carbon atoms optionallysubstituted at one or more atoms. Optionally, L comprises a(CH₂—CH₂—O—)_(x) group, wherein x is an integer from among the range of1 to 10. Optionally, the groups —(C)_(n)—X-L— collectively comprise astructure (CH₂—CH₂—O—)_(x), wherein x is an integer from among the rangeof 2 to 20, optionally wherein x is an integer from among the range of 3to 24. Optionally, L comprises an amino acid or a di-, tri-, tetra-, oroligopeptide. In some embodiments, L is alanine, arginine, asparagine,aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine, valine, or citrulline. In someembodiments, L is valine or citrulline.

Optionally, in any of the linking reagents, protein-conjugated linkingreagents, antibodies or antibody fragments of the invention, theacceptor glutamine residue(s) is/are flanked at the +2 position by anon-aspartic acid residue. Optionally, the residue at the +2 position isin the Fc domain of a heavy chain, optionally at position 297 (EU indexnumbering) of a heach chain. In one embodiment, the residue at the +2position is a non-aspartic acid, non-glutamine residue. In oneembodiment, the residue at the +2 position is a non-aspartic acid,non-asparagine residue. In one embodiment, the residue at the +2position is a non-negatively charged amino acid. In one embodiment, theresidue at the +2 position is a serine or threonine. In one embodiment,an antibody of Formula IVa comprises one acceptor glutamine on eachheavy chain.

In one embodiment, the present invention relates to a method forconjugating a moiety of interest to an antibody, comprising the stepsof:

a) providing an antibody having at least one acceptor glutamine residue,optionally an acceptor glutamine residue flanked at the +2 position by anon-aspartic acid residue;

b) reacting said antibody with a lysine-based linker of Formula Ia, inthe presence of a TGase, under conditions sufficient to obtain anantibody of Formula IVa.

Linking reagents used for multi-step linking of a moiety of interest (Z)to an antibody will advantageously comprise one or more reactive groups.A reactive group can be a thiol, a maleimide, a halo-acetamide (e.g.bromo-acetamide, iodo-acetamide, cloro-acetamide) an o-phoshenearomaticester, an azide, a fulminate, an alkyne, a cyanide, an anthracene, adiene, a 1,2,4,5-tetrazine, or a norbornene, a cylcooctyne (e.g. adibenzocyclooctyne) or other strained cycloalkene, where two or morecompatible reactive groups can be attached to the linking reagent. Thereactive group of the linking reagent is chosen to undergothio-maleimide (or halo-acetamide) addition, Staudinger ligation,Huisgen 1,3-cycloaddition (click reaction), or Diels-Alder cycloadditionwith a complementary reactive group attached to an agent comprising atherapeutic moiety, a diagnostic moiety, or any other moiety for adesired function.

In one embodiment, the invention provides a linking reagent, apharmaceutically acceptable salt or solvate thereof, or aprotein-conjugated linking reagent having the general Formula Ib:

G-NH—(C)_(n)—X-L-(V—(Y—(R)_(z))_(q))_(r)  Formula Ib

or a pharmaceutically acceptable salt or solvate thereof,

wherein:

G is an H, amine protecting group, an antibody or antibody fragment orother protein attached via an amide bond (e.g. through an acceptorglutamine residue in the primary sequence of the antibody or antibodyfragment);

(C)_(n) is a substituted or unsubstituted alkyl or heteroalkyl chain,optionally wherein any carbon of the chain is substituted with analkoxy, hydroxyl, alkylcarbonyloxy, alkyl-S—, thiol, alkyl-C(O)S—,amine, alkylamine, amide, or alkylamide;

n, is an integer from among the range of 2 to 20 atoms, preferably 3 to6;

X is NH, O, S, or absent;

L is a bond or a carbon comprising framework of 1 to 200 atomssubstituted at one or more atoms, optionally wherein the carboncomprising framework is a linear hydrocarbon, a symmetrically orasymmetrically branched hydrocarbon, monosaccharide, disaccharide,linear or branched oligosaccharide (asymmetrically branched orsymmetrically branched), other natural linear or branched oligomers(asymmetrically branched or symmetrically branched), or a dimer, trimer,or higher oligomer (linear, asymmetrically branched or symmetricallybranched) resulting from any chain-growth or step-growth polymerizationprocess;

r is an integer selected from among 1, 2, 3 or 4;

q is an integer selected from among 1, 2, 3 or 4; and

z is an integer selected from among 1, 2, 3 or 4;

V is independently absent, a bond or a continuation of a bond if L is abond, a non-cleavable moiety or a conditionally-cleavable moiety thatcan optionally be cleaved or transformed by a chemical, photochemical,physical, biological, or enzymatic process (e.g. cleavage of Vultimately leading to release of one or more moieties subsequently orultimately linked to V, for example a Z moiety). In some embodiments, Vis, preferably, a di-, tri-, tetra-, or oligopeptide as described belowin the section entitled “The V Moiety”;

Y is independently absent, a bond or a continuation of a bond, or aspacer system (e.g., a self-eliminating spacer system or anon-self-elimination spacer system) which is comprised of 1 or morespacers; and

R is a reactive moiety, preferably a moiety comprising an unprotected orprotected thiol, maleimide, haloacetamide, o-phoshenearomatic ester,azide, fulminate, alkyne, cyanide, anthracene, 1,2,4,5-tetrazine,norbornene, other stained or otherwise electronically activated alkeneor, optionally, a protected or unprotected amine when X is absent and L,V, or Y is other than a bond or a continuation of a bond. In analternative embodiment R is a reactive moiety, preferably a moietycomprising an unprotected or protected thiol, an unprotected orprotected amine, maleimide, haloacetamide, o-phoshenearomatic ester,azide, fulminate, alkyne, cyanide, anthracene, 1,2,4,5-tetrazine,norbornene, other stained or otherwise electronically activated alkene,provided that R is not an amine when n=5 and X, L, V and Y are absent.Optionally, R is not an amine when n=4 and X, L, V and Y are absent.

In one embodiment, the present invention relates to a method forconjugating a moiety of interest to an antibody, comprising the stepsof:

a) providing an antibody having at least one acceptor glutamine residue;

b) reacting said antibody with a lysine-based linker of Formula Ib, inthe presence of a TGase, under conditions sufficient to obtain anantibody of Formula II.

The method optionally further comprises evaluating the stoichiometry ofthe conjugated antibodies (e.g., the ratio of functionalized acceptorglutamines:antibody) by mass spectrometry, preferably liquidchromatography mass spectrometry (LC/MS) or hydrophobic interactionchromatography (HIC).

In one embodiment, the invention provides an antibody, wherein anantibody (Ab) comprises an acceptor glutamine residue conjugated (i.e.,covalently attached) to one or more reactive moieties (R) through alinker that comprises a —NH—(C)_(n)—X moiety. In one embodiment, theinvention provides an antibody or antibody fragment comprising afunctionalized acceptor glutamine residue having Formula II:

(Q)-NH—(C)_(n)—X-L-(V—(Y—(R)_(z))_(q))_(r)  Formula II

or a pharmaceutically acceptable salt or solvate thereof,

wherein:

Q is glutamine residue present in an antibody or antibody fragment;

(C)_(n) is a substituted or unsubstituted alkyl or heteroalkyl chain,optionally wherein any carbon of the chain is substituted with analkoxy, hydroxyl, alkylcarbonyloxy, alkyl-S—, thiol, alkyl-C(O)S—,amine, alkylamine, amide, or alkylamide;

n is an integer from among the range of 2 to 20;

X is NH, O, S, or absent;

L is a bond or a carbon comprising framework of 1 to 200 atomssubstituted at one or more atoms, optionally wherein the carboncomprising framework is a linear hydrocarbon, a symmetrically orasymmetrically branched hydrocarbon monosaccharide, disaccharide, linearor branched oligosaccharide (asymmetrically branched or symmetricallybranched), other natural linear or branched oligomers (asymmetricallybranched or symmetrically branched), or a dimer, trimer, or higheroligomer (linear, asymmetrically branched or symmetrically branched)resulting from any chain-growth or step-growth polymerization process;

r is an integer selected from among 1, 2, 3 or 4;

q is an integer selected from among 1, 2, 3 or 4;

z is an integer selected from among 1, 2, 3 or 4; and

V is independently absent, a bond or a continuation of a bond, anon-cleavable moiety or a conditionally-cleavable moiety that canoptionally be cleaved or transformed by a chemical, photochemical,physical, biological, or enzymatic process (e.g. cleavage of Vultimately leading to release of one or more moieties subsequently orultimately linked to V, for example a Z moiety). In some embodiments, Vis, preferably, a di-, tri-, tetra-, or oligopeptide as described belowin the section entitled “The V Moiety”;

Y is independently absent, a bond or a continuation of a bond, or aspacer system (e.g., a self-eliminating spacer system or anon-self-elimination spacer system) which is comprised of 1 or morespacers; and

R is a reactive moiety, preferably a moiety comprising an unprotected orprotected thiol, maleimide, haloacetamide, o-phoshenearomatic ester,azide, fulminate, alkyne, cyanide, anthracene, 1,2,4,5-tetrazine,norbornene, other stained or otherwise electronically activated alkeneor, optionally, a protected or unprotected amine when X is absent and L,V, or Y is other than a bond or a continuation of a bond. In analternative embodiment R can be a reactive moiety, preferably a moietycomprising an unprotected or protected thiol, an unprotected orprotected amine, maleimide, haloacetamide, o-phoshenearomatic ester,azide, fulminate, alkyne, cyanide, anthracene, 1,2,4,5-tetrazine,norbornene, other stained or otherwise electronically activated alkene,provided that R is not an amine when n=5 and X, L, V and Y are absent.Optionally, R is not an amine when n=4 and X, L, V and Y are absent.

In one embodiment of the methods, the antibody of Formula II is reactedwith a compound comprising a moiety-of-interest Z and a reactive group(R′) complementary for forming at least one bond with reactive group Rof Formula Ib or II. Optionally, the moiety of interest is a therapeuticor diagnostic moiety (Z). In one aspect, the compound comprising amoiety-of-interest Z and a reactive group (R′) comprises a structure ofFormula III, below,

R′-L′-(V′—(Y′—(Z or R)_(z′))_(q′))_(r′)  Formula III

or a pharmaceutically acceptable salt or solvate thereof,

wherein:

R′ is a reactive group, e.g. a reactive group complementary for formingat least one bond with reactive group R of Formula Ib, Ic or II:

G-NH—(C)_(n)—X-L-(V—(Y—(R)_(z))_(q))_(r)  Formula Ib

G-NH—(C)_(n)—X-L-(V—(Y-(M)_(z))_(q))_(r)  Formula Ic

(Q)-NH—(C)_(n)—X-L-(V—(Y—(R)_(z))_(q))_(r)  Formula II;

L′ is a bond or a carbon comprising framework of 1 to 200 atomssubstituted at one or more atoms, optionally wherein the carboncomprising framework is a linear hydrocarbon, a symmetrically orasymmetrically branched hydrocarbon monosaccharide, disaccharide, linearor branched oligosaccharide (asymmetrically branched or symmetricallybranched), other natural linear or branched oligomers (asymmetricallybranched or symmetrically branched), or a dimer, trimer, or higheroligomer (linear, asymmetrically branched or symmetrically branched)resulting from any chain-growth or step-growth polymerization process;

V′ is independently absent, a non-cleavable moiety or aconditionally-cleavable moiety that can optionally be cleaved ortransformed by a chemical, photochemical, physical, biological, orenzymatic process, cleavage of V ultimately leading to release of one ormore Z moieties. In some embodiments, V is, preferably, a di-, tri-,tetra-, or oligopeptide as described below in the section entitled “TheV Moiety”,

Y′ is independently absent, a bond or a continuation of a bond, or aspacer system (e.g., a self-eliminating spacer system or anon-self-elimination spacer system) which is comprised of 1 or morespacers;

Z is independently a moiety that improves the pharmacokineticproperties, a therapeutic moiety, or diagnostic moiety;

R is a reactive group (optionally protected) other than a complementaryreactive group for reaction with R′, optionally a moiety comprising anunprotected or protected thiol, an unprotected or protected amine,maleimide, haloacetamide, o-phoshenearomatic ester, azide, fulminate,alkyne, cyanide, anthracene, 1,2,4,5-tetrazine, norbornene, otherstained or otherwise electronically activated alkene,

q′ and r′ are an integer selected from among 1, 2, 3 or 4; and

z′ is an integer selected from among 1, 2, 3 or 4.

The compound of Formula Ib can optionally be reacted with a reactionpartner (e.g. a compound of Formula III) to create pre-assembled linkerintermediates. The invention thus also provides a functionalizedlysine-based linker of Formula Ic:

G-NH—(C)_(n)—X-L-(V—(Y-(M)_(z))_(q))_(r)  Formula Ic

wherein each of G, C, n, X, L, V, Y, z, q, and r are as defined inFormula Ia, and M is independently: R or(RR′)-L′-(V′—(Y′—(Z)_(z′))_(q′))_(r′), wherein each of L′, V′, Y′, z′,q′, and r′ are as defined in Formula III, R is as defined in Formula Iand wherein each (RR′) is an addition product between an R of Formula Iand its complementary R′ of formula III (see, for example, FIG. 1 andFIG. 2). Thus, RR′ can be an addition product of a: thio-maleimide (orhaloacetamide) addition, for example, aN,S-disubstituted-3-thio-pyrrolidine-2,5-dione; Staudinger ligation, forexample, a N,3- or N,4-substitued-5-dipenylphosphinoxide-benzoic amide;Huisgen 1,3-cycloaddition (click reaction), for example, a N,S-disubstituted-3-thio-pyrrolidine-2,5-dione,1,4-disubstituted-1,2,3-triazole, 3,5-disubstituted-isooxazole, or3,5-disubstituted-tetrazole; Diels-Alder cycloaddition adduct, forexample the 2,4-cycloaddition product between an O orN-substituted-5-norbornene-2-carboxylic ester or amide,N-substituted-5-norbornene-2,3-dicarboxylic imide, O orN-substituted-7-oxonorbornene-5-carboxylic ester or amide, orN-substituted-7-oxonorbornene-5,6-dicarboxylic imide and a 9-substitutedanthracene or 3-substituted 1,2,4,5-tetrazine; or any high yieldselective amidation or imidization reaction. Some reactions and thecorresponding RR′ reaction products are illustrated in FIGS. 1 and 2.

Optionally, a compound will comprise V or V′ (but not both V and V′).Optionally, a compound will comprise Y or Y′ (but not both Y and Y′).

The linkers of Formula Ib can be reacted with an antibody, in thepresence of a TGase and under suitable conditions, to produce anantibody comprising a functionalized acceptor glutamine of Formula IVb.

In one embodiment, the invention provides an antibody or antibodyfragment comprising a functionalized acceptor glutamine residue havingFormula IVb, below,

(Q)-NH—(C)_(n)—X-L-(V—(Y-(M)_(z))_(q))_(r)  Formula IVb

or a pharmaceutically acceptable salt or solvate thereof,

wherein:

Q is glutamine residue present in an antibody or antibody fragment;

(C)_(n) is a substituted or unsubstituted alkyl or heteroalkyl chain,optionally wherein any carbon of the chain is substituted with analkoxy, hydroxyl, alkylcarbonyloxy, alkyl-S—, thiol, alkyl-C(O)S—,amine, alkylamine, amide, or alkylamide;

n is an integer from among the range of 2 to 20;

X is NH, O, S, or absent;

L is a bond or a carbon comprising framework of 1 to 200 atomssubstituted at one or more atoms, optionally wherein the carboncomprising framework is a linear hydrocarbon, a symmetrically orasymmetrically branched hydrocarbon monosaccharide, disaccharide, linearor branched oligosaccharide (asymmetrically branched or symmetricallybranched), other natural linear or branched oligomers (asymmetricallybranched or symmetrically branched), or a dimer, trimer, or higheroligomer (linear, asymmetrically branched or symmetrically branched)resulting from any chain-growth or step-growth polymerization process;

r is an integer selected from among 1, 2, 3 or 4;

q is an integer selected from among 1, 2, 3 or 4;

z is an integer selected from among 1, 2, 3 or 4; and

V is independently absent, a bond or a continuation of a bond or anon-cleavable moiety or a conditionally-cleavable moiety that canoptionally be cleaved or transformed by a chemical, photochemical,physical, biological, or enzymatic process (e.g. cleavage of Vultimately leading to release of one or more moieties subsequently orultimately linked to V, for example a Z moiety). In some embodiments, Vis, preferably, a di-, tri-, tetra-, or oligopeptide as described belowin the section entitled “The V Moiety”;

Y is independently absent, a bond or a continuation of a bond, or aspacer system (e.g., a self-eliminating spacer system or anon-self-elimination spacer system) which is comprised of 1 or morespacers; and

Z is a moiety-of-interest, optionally a moiety that improves thepharmacokinetic properties, or a therapeutic moiety or a diagnosticmoiety, and each Z is directly coupled to either Y or V when Y isabsent, or L when both Y and V are absent.

M is independently: R or (RR′)-L′-(V′—(Y′—(Z)_(z′))_(q′))_(r′), whereineach of L′, V′, Y′, z′, q′, and r′ are as defined in Formula III for L,V, Y, z, q, and r, Z is a moiety-of-interest, optionally a moiety thatimproves the pharmacokinetic properties, or a therapeutic moiety or adiagnostic moiety, R is as defined in Formula I and wherein each (RR′)is an addition product between an R of Formula I and its complementaryR′ of formula III (see, for example, FIG. 1 and FIG. 2). RR′ ispreferably an addition product of a: thio-maleimide (or haloacetamide)addition, for example, a N,S-disubstituted-3-thio-pyrrolidine-2,5-dione;Staudinger ligation, for example, a N,3- orN,4-substitued-5-dipenylphosphinoxide-benzoic amide; Huisgen1,3-cycloaddition (click reaction), for example, aN,S-disubstituted-3-thio-pyrrolidine-2,5-dione,1,4-disubstituted-1,2,3-triazole, 3,5-disubstituted-isooxazole, or3,5-disubstituted-tetrazole; Diels-Alder cycloaddition adduct, forexample the 2,4-cycloaddition product between an O orN-substituted-5-norbornene-2-carboxylic ester or amide,N-substituted-5-norbornene-2,3-dicarboxylic imide, O orN-substituted-7-oxonorbomene-5-carboxylic ester or amide, orN-substituted-7-oxonorbornene-5,6-dicarboxylic imide and a 9-substitutedanthracene or 3-substituted 1,2,4,5-tetrazine; or any high yieldselective amidation or imidization reaction. Some reactions and thecorresponding RR′ reaction products are illustrated in FIGS. 1 and 2.

Optionally, Formula IVb will comprise V or V′ (but not both V and V′).Optionally, Formula IV will comprise Y or Y′ (but not both Y and Y′).

The present invention also relates to a method for conjugating a moietyof interest to an antibody, comprising the steps of:

a) providing an antibody comprising at least one acceptor glutamineresidue; and

b) reacting said antibody with a lysine-based linker of Formula Ib orIc, in the presence of a TGase, under conditions sufficient to obtain anantibody comprising a functionalized acceptor glutamine residue (Q) ofFormula II or IVb, respectively;

c) optionally, reacting the antibody-lysine-based linker conjugate ofFormula II of step (b), optionally immobilized on a solid support, witha compound of Formula III to obtain an antibody with a moiety ofinterest Z covalently bound thereto (e.g., via —NH—(C)_(n)—X-L andoptionally further via V, and/or Y). For example, an antibody comprisinga functionalized acceptor glutamine residue (Q) of Formula IVb, below,is obtained.

In another aspect, the present invention thus relates to a method forconjugating a moiety (Z) of interest to an antibody, comprising thesteps of:

a) providing an antibody having at least one acceptor glutamine residue;and

b) reacting said antibody with a lysine-based linker (e.g. a linker ofFormula Ia, Ib or Ic), in the presence of a TGase, under conditionssufficient to obtain an antibody of Formula II, IVa or IVb, wherein saidlysine-based linker is provided at between 2 and 80 (or between 4 and80) molar equivalents to the antibody, optionally between 2 and 20 (orbetween 4 and 20, between 4 and 40, or between 2 and 40) molarequivalents to the antibody, optionally at a less than 80, 40, 20 or 10molar equivalents to the antibody.

In another aspect, the present invention thus relates to a method forconjugating a moiety (Z) of interest to an antibody, comprising thesteps of:

a) providing an antibody comprising at least one acceptor glutamineresidue;

b) reacting said antibody with a lysine-based linker (e g a linker ofFormula Ib or Ic), in the presence of a TGase, under conditionssufficient to obtain an antibody comprising a functionalized acceptorglutamine residue (Q) of Formula II, wherein said lysine-based linker isprovided at between 2 and 80 (or between 4 and 80) molar equivalents tothe antibody, optionally between 2 and 20 (or between 4 and 20, between4 and 40, or between 2 and 40) molar equivalents to the antibody,optionally at a less than 80, 40, 20 or 10 molar equivalents to theantibody; and

c) reacting a sample of an antibody comprising a functionalized acceptorglutamine residue of Formula II of step b), optionally wherein theantibody is immobilized on a solid support, with a compound of FormulaIII, optionally in aqueous conditions, to obtain an antibody compositionof Formula IVb comprising a Z moiety, wherein said compound of FormulaIII is provided between 2 and 80 molar equivalents to the antibody,optionally between 2 and 20 or between 2 and 40 molar equivalents to theantibody, optionally at a less than 80, 40, 20, 10, 5, 4 or 3 molarequivalents to the antibody, e.g. for an antibody comprising oneacceptor glutamine on each heavy chain. In one embodiment, said compoundof Formula III is provided between 4 and 80 molar equivalents to theantibody, optionally between 4 and 20 or between 4 and 40 molarequivalents to the antibody, optionally at a less than 80, 40, 20, 10 or5 molar equivalents to the antibody, e.g. for an antibody comprising twoacceptor glutamines on each heavy chain.

Optionally, step (c) comprises: immobilizing a sample of an antibodycomprising a functionalized acceptor glutamine residue of Formula II ona solid support to provide a sample comprising immobilized antibodies,reacting the sample comprising immobilized antibodies with a compound ofFormula III, optionally recovering any unreacted compound andre-introducing such recovered compound to the solid support for reactionwith immobilized antibodies, and eluting the antibody conjugates toprovide an antibody composition of Formula IVb comprising a Z moiety.

In one embodiment, the invention relates to a method for evaluating aplurality of Z and/or R moieties (e.g. a method of screening drugsand/or linkers for suitability for use as an antibody-drug conjugate),the method comprising the steps of:

a) providing a first and a second sample of an antibody comprising afunctionalized acceptor glutamine residue of Formula II, preferablywherein said samples are obtained by reacting an antibody with alysine-based linker (e.g. a linker of Formula Ib), in the presence of aTGase, under conditions sufficient to obtain an antibody comprising afunctionalized acceptor glutamine residue (Q) of Formula II;

b) reacting the first sample of an antibody comprising a functionalizedacceptor glutamine residue of Formula II of step a), optionallyimmobilized on a solid support, with a compound of Formula III to obtaina first antibody composition of Formula IVb comprising a first Z moiety,and

c) reacting the second sample of antibody comprising a functionalizedacceptor glutamine residue of step b), optionally immobilized on a solidsupport, with a compound of Formula III that differs in its Z moietyfrom the compound of Formula III or step (c), to obtain a secondantibody composition of Formula IVb comprising a second Z moiety.

Optionally, steps (b) and (c) comprise: immobilizing a sample of saidfirst or second antibody comprising a functionalized acceptor glutamineresidue of Formula II on a solid support to provide a sample comprisingimmobilized antibodies, reacting the sample comprising immobilizedantibodies with a compound of Formula III, optionally recovering anyunreacted compound and re-introducing such recovered compound to thesolid support for reaction with immobilized antibodies, and eluting theantibody conjugates to provide a first or second antibody composition ofFormula IVb comprising a first or second Z moiety, respectively.

In one embodiment, the invention relates to a method for evaluating aplurality of Z and/or R moieties (e.g. a method of screening drugsand/or linkers for suitability for use as an antibody-drug conjugate),the method comprising the steps of:

a) providing an antibody comprising at least one acceptor glutamineresidue;

b) reacting said antibody with a lysine-based linker (e g a linker ofFormula Ib or Ic), in the presence of a TGase, under conditionssufficient to obtain an antibody comprising a functionalized acceptorglutamine residue (Q) of Formula II;

c) reacting a first sample of an antibody comprising a functionalizedacceptor glutamine residue of Formula II of step b), optionallyimmobilized on a solid support, with a compound of Formula III to obtaina first antibody composition of Formula IVb comprising a first Z moiety,and

d) reacting a second sample of antibody comprising a functionalizedacceptor glutamine residue of step b), optionally immobilized on a solidsupport, with a compound of Formula III that differs in its Z moietyfrom the compound of Formula III or step (c), to obtain a secondantibody composition of Formula IVb comprising a second Z moiety.

Optionally, steps (c) and (d) comprise: immobilizing a said first orsecond antibody comprising a functionalized acceptor glutamine residueof Formula II on a solid support to provide a sample comprisingimmobilized antibodies, reacting the sample comprising immobilizedantibodies with a compound of Formula III, optionally recovering anyunreacted compound and re-introducing such recovered compound to thesolid support for reaction with immobilized antibodies, and eluting theantibody conjugates to provide a first or second antibody composition ofFormula IV comprising a first or second Z moiety, respectively.

The L, V, Y, L′, V′, Y′ groups of IV can be like or different dependingon the structure of Formula II and/or Formula III used, as desired.

Optionally the method further comprises a step e) evaluating eachantibody conjugate of (b) and (c) (or (c) and (d)), e.g. for biologicalactivity, any physical or pharmacokinetic properties. Optionally, thestep of evaluating comprises comparing a first antibody to a secondantibody for biological activity, pharmacokinetic properties. In oneembodiment of the above method, the antibody comprising a functionalizedacceptor glutamine residue of Formula II has acceptor glutamine residuehaving a structure of column “Formula II” of Table 1, row n, and thecompound of Formula III has a structure of column “Formula II” of Table1, row n, wherein n=1-10. Optionally, the step of evaluation an antibodyconjugate comprises the method of the respective row in Table 1, column“Exemplary evaluation method”.

Optionally any of the methods further comprise a step of evaluating thestoichiometry of the conjugated antibodies (e.g., the ratio offunctionalized acceptor glutamines:antibody) by analyticalchromotagraphic methods and/or mass spectrometry. In one embodiment,liquid chromatography mass spectrometry (LC/MS) is used. In oneembodiment, hydrophobic interaction chromatography is used.

The invention also provides a method comprising reacting afunctionalized lysine derivative of Formula II with a compoundcomprising a moiety of interest Z and a reactive group (R′) capable offorming at least one bond with reactive group R of Formula Ib, Ic or II(e.g. a compound of Formula III).

In one embodiment, the invention relates to a method for evaluating anantibody conjugate, or evaluating one or more Z moieties that are linkedand/or combined in different manners (e.g. different L, V, Y, L′, V′,Y′, r, q, z, r′, q′ and/or z′ (e.g. a method of screening drugs,diagnostic moieties, moieties that improve pharmacokinetic properties,and/or linkers for suitability for use as an antibody-drug conjugate),the method comprising the steps of:

a) providing a first antibody composition of Formula IVb comprising afirst X, L, V, Y, L′, V′, Y′, (RR′) and/or Z moiety, wherein at least80%, 90%, 95%, 98% or 99% of the antibodies in said first antibodycomposition have (m) functionalized acceptor glutamine residues (Q) perantibody, e.g. wherein m=1, 2 or 4,

b) providing a second antibody composition of Formula IVb comprising asecond X, L, V, Y, L′, V′, Y′, (RR′) and/or Z moiety, wherein saidsecond antibody comprises at least one X, L, V, Y, L′, V′, Y′, (RR′)and/or Z moiety that differs from a respective X, L, V, Y, L′, V′, Y′,(RR′) and/or Z moiety of said first antibody (e.g., a moiety differs instructure or by being present or absent), wherein at least 80%, 90%,95%, 98% or 99% of the antibodies in said second antibody compositionhave (n) functionalized acceptor glutamine residues (Q) per antibody;and

c) evaluating the first and second antibody compositions, e.g. forbiological activity, any physical or pharmacokinetic properties.Optionally the first and second antibody composition is a compositionmade according to any of the methods of the invention. In oneembodiment, n and m are equal. Preferably, m and/or n are 1, 2 or 4. Inone embodiment, the antibodies of the first and second antibodycompositions are directed to the same predetermined antigen. In oneembodiment, the antibodies of the first and second antibody compositionsshare heavy and/or light chain amino acid sequences. In one embodiment,the antibodies of the first and second antibody compositions differ intheir heavy and/or light chain amino acid sequences.

When more than one R group is present in a compound of the formulaeherein (e.g. Formula Ib, Ic, II, III or IVb), the R groups willpreferably be compatible such that no R group is a complementary reagentto any other R group. The L group can be a carbon comprising framework,where L is a linear hydrocarbon, a symmetrically or asymmetricallybranched hydrocarbon, monosaccharide, disaccharide, linear or branchedoligosaccharide (asymmetrically branched or symmetrically branched),other natural oligomer, dimer, trimer, or higher oligomer (linearasymmetrically branched or symmetrically branched) resulting from anychain-growth or step-growth polymerization process, wherein L has r, q,and/or z sites of attachment for the respective V, Y, and R groups,where r and q represent the degree of branching or polymerization. Thesites of attachment can comprise a bond or comprise a functional groupselected from an alkene, alkyne, ether, thioether, ester, thioester,amine, amide, alkylamide, or other functional group readily generated bya condensation or addition reaction. An example of amulti-functionalized linking reagent where r=3 is shown in FIG. 9.

When more than one Z group is present in a compound of the formulaeherein (e.g. Formula Ia, III or IVa), the L group can be a carboncomprising framework, where L is a linear hydrocarbon, a symmetricallyor asymmetrically branched hydrocarbon, monosaccharide, disaccharide,linear or branched oligosaccharide (asymmetrically branched orsymmetrically branched), other natural oligomer, dimer, trimer, orhigher oligomer (linear asymmetrically branched or symmetricallybranched) resulting from any chain-growth or step-growth polymerizationprocess, wherein L has r, q, and/or z sites of attachment for therespective V, Y, and Z groups, where r and q represent the degree ofbranching or polymerization. The sites of attachment can comprise a bondor comprise a functional group selected from an alkene, alkyne, ether,thioether, ester, thioester, amine, amide, alkylamide, or otherfunctional group readily generated by a condensation or additionreaction. An example of a multi-functionalized linking reagent where r=3is shown in FIG. 9.

The antibody or antibody fragment according the any of the methods andcompositions according to the invention will preferably provide at leastone acceptor glutamine residue (e.g. one or two acceptor glutamineresidues per heavy chain of the antibody). The lysine-basedlinker-derivatized moiety-of-interest, when attached to an antibody,shall preferably comprise a low-molecular mass primary amine, optionallyan amino pentyl, optionally a 5-amino pentyl residue, and themoiety-of-interest. Optionally, —NH—(C)_(n)—X can be specified to be anysuitable low-molecular mass primary amine, optionally a 5-amino pentylresidue, respectively that is recognized by TGase, e.g., which allowsthe selective formation of covalently linked conjugates between theγ-carboxamide groups of glutamine and a free pendent primary amino groupof the lysine-derivatized drug. The method allows the selectiveformation of covalently linked conjugates between the γ-carboxamidegroups of glutamine and a free pendent primary amino group of thelysine-based linker.

As presented herein, the acceptor glutamine residue is part of theimmunoglobulin and the low-molecular mass primary amine, optionally anaminopentyl, optionally a 5-amino pentyl residue is part of thelysine-based linker moiety that is conjugated to the glutamine residueon the immunoglobulin.

In one embodiment, the invention provides an antibody (Ab) comprising anacceptor glutamine residue (Q) conjugated (i.e., covalently attached)via said acceptor glutamine residue (Q) to one or moremoieties-of-interest (Z) through a linker that comprises a —NH—(C)_(n)—Xmoiety, optionally wherein the linker further comprises a (RR′) moiety.The optional RR′ moiety may be, for example, an addition product of a:thio-maleimide (or haloacetamide) addition, for example, aN,S-disubstituted-3-thio-pyrrolidine-2,5-dione; Staudinger ligation, forexample, a N,3- or N,4-substitued-5-dipenylphosphinoxide-benzoic amide;Huisgen 1,3-cycloaddition (click reaction), for example, aN,S-disubstituted-3-thio-pyrrolidine-2,5-dione,1,4-disubstituted-1,2,3-triazole, 3,5-disubstituted-isooxazole, or3,5-disubstituted-tetrazole; Diels-Alder cycloaddition adduct, forexample the 2,4-cycloaddition product between an O orN-substituted-5-norbornene-2-carboxylic ester or amide,N-substituted-5-norbornene-2,3-dicarboxylic imide, O orN-substituted-7-oxonorbornene-5-carboxylic ester or amide, orN-substituted-7-oxonorbornene-5,6-dicarboxylic imide and a 9-substitutedanthracene or 3-substituted 1,2,4,5-tetrazine; or any high yieldselective amidation or imidization reaction.

It will be appreciated that Formula II and IVa and IVb can forconvenience also be expressed as(Ab)-NH—(C)_(n)—X-L-(V—(Y—(R)_(z))_(q))_(r),(Ab)-NH—(C)_(n)—X-L-(V—(Y—(Z)_(z))_(q))_(r) and(Ab)-NH—(C)_(n)—X-L-(V—(Y-(M)z)_(q))_(r), respectively, where (Ab) is animmunoglobulin (Ab) is conjugated via a glutamine (Q) residue to an NHof the linking reagent (e.g. the compound of Formula Ia, Ib or Ic).

In any of Formulas I to IV, q, q′, r and r′ may optionally be specifiedto represent degree of branching or polymerization.

In Formula IVa or IVb, the total number of R or Z moieties per antibodyis preferably from about 1 to about 16. The invention includes acomposition comprising a plurality of antibody compounds of Formula IVaor IVb, wherein substantially each antibody of such plurality has 1, 2,3, 4, 5, 6, 8, 10, 12, 14 or 16 moieties Z per antibody.

In one embodiment, the antibody of Formulae II, IVa or IVb has one, twoor four functionalized acceptor glutamine residue and z=1, q=1 and r=1.In one embodiment, the antibody of Formulae II, IVa or IVb has one, twoor four functionalized acceptor glutamine residues and z=2, 3 or 4, q=1and r=1. In one embodiment, the antibody of Formulae II, IVa or IVb hasone, two or four functionalized acceptor glutamine residues and z=1, q=2and r=1. In one embodiment, the antibody of Formula IVa has one, two orfour functionalized acceptor glutamine residues and z=1, q=1 and r=2, 3or 4. In one embodiment, the antibody of Formulae II, IVa or IVb hasone, two or four functionalized acceptor glutamine residues and z=1, q=1and r=1. In one embodiment invention provides an antibody composition inwhich Z (e.g. drug) loading per antibody is homogeneous, optionally atleast 70%, 80% or 90% of the antibodies in a composition have the samenumber of Z moieties per antibody.

In any of the embodiments herein, the moiety of interest Z may be atherapeutic moiety, a diagnostic moiety or a moiety that improves thepharmacokinetic properties of the compound. In any embodiment herein,particularly when Z or R is an organic compound that is electronicallynegatively charge, hydrophobic, rigid (e.g. presence or a cyclic group,a polycycle, a macrocycle), and/or has a molecular weight of at least400 g/mol, the compounds of Formulae Ia, Ib, Ic, II, III, IVa or IVb maycomprise a group (C)_(n), L, L′, V, V′, Y and/or Y′ that acts as aspacer to distance the moiety-of-interest (Z) from the primary amine(and thus reactive site of TGase).

In one embodiment, the (C)_(n) group, optionally together with othermoieties (e.g. L, L′, V, V′, Y and/or Y′) serves as a spacer, wherein nis an integer greater than 6, optionally wherein n is an integer greaterthan 10). Optionally, the (C)_(n) group comprises a (CH₂CH₂O)_(x) groupwhere x is an integer selected among the range of 1 to 6, optionallywhere x is an integer selected among the range of 1 to 10.

In one embodiment, said the L group is present in a compound of theinvention (is not a bond) and serves as a spacer, optionally wherein theL group comprises or consists of a carbon-comprising framework of:

a) 2-15 linear carbon atoms optionally substituted at one or more atoms;

b) 2-15 linear carbon atoms optionally substituted at one or more atoms;

c) 5-20 linear carbon atoms optionally substituted at one or more atoms;

d) 6-30 linear carbon atoms optionally substituted at one or more atoms;

e) 5-15 linear carbon atoms optionally substituted at one or more atoms;or

f) 4, 5 or 6 linear carbon atoms optionally substituted at one or moreatoms.

In some embodiments, L is alanine, arginine, asparagine, aspartic acid,cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, valine or citrulline. In some embodiments, L isvaline or citrulline. In one embodiment, the L group comprises a(CH₂CH₂O)_(x) group where x is an integer selected among the range of 1to 6, optionally where x is an integer selected among the range of 1 to10.

In one embodiment, the V group is present in a compound of the invention(is not a bond) and serves as a spacer, optionally wherein the V groupcomprises a di-, tri-, tetra, or oligopeptide(s).

The moiety that serves as a spacer may also comprise or consist of anycombination of the (C)_(n), L, V or Y groups (e.g. (C)_(n) and L, V orY; (C)_(n), L, V and Y; (C)_(n), V and Y; (C)_(n), L and Y; (C)_(n), Land V).

In any of the compounds of the invention (e.g. in any of Formula I, IIand/or IV), each of (C)_(n), the linking element (L), the V moiety orthe Y moiety can optionally be characterized as having a chain length ofat least 2.8 Angstroms, 3 Angstroms, 4 Angstroms, 5 Angstroms, 10Angstroms, 15 Angstroms or 18 Angstroms. Optionally, the chain lengthcan be characterized by an upper range of 200, 100 or 50 Angstroms.

In one embodiment of any of the compounds of the invention (e.g. in anyof Formula I, II and/or IV), the combination of (C)_(n), linking element(L), the V moiety and the Y moiety, or subcombinations thereof (e.g.,(C)_(n) and L, L, V and Y, L and Y, L and V, V and Y), can optionally becharacterized as collectively having a chain length of at least 2.8Angstroms, 3 Angstroms, 4 Angstroms, 5 Angstroms, 10 Angstroms, 15Angstroms or 18 Angstroms. Optionally, the chain length can becharacterized by an upper range of 200, 100 or 50 Angstroms.

In any of the compounds of the invention, a compound of Formula Ia, Ib,Ic, II, IVb and/or IVb can optionally be characterized as comprising asingle lysine or functional equivalent group, e.g. a singleTGase-accessible primary amine or NH—(C)_(n)— group.

In any the methods or compositions of the invention, a composition of aplurality of antibody conjugates is obtained wherein substantially all(e.g. at least 90%, 95%, 98% or 99%) the antibodies in the compositioncomprise a functionalized acceptor amino acid (e.g. glutamine) on aheavy chain constant region (e.g. on a CH2 domain) Preferably, in anythe methods or compositions of the invention, a composition of aplurality of antibody conjugates is obtained wherein the antibodies havea homogeneous ratio of functionalized acceptor amino acids (e.g.glutamine):antibody. In one embodiment the invention provides acomposition comprising a plurality of antibodies of Formula II or IV,wherein at least 70%, 80%. 85%, 90%, 95%, 98% or 99% of the antibodiesin the composition have the same number of functionalized acceptorglutamine residues (Q) (e.g., a functionalized acceptor glutamine ofFormula II, IVa or IVb) per antibody. Preferably at least 70%, 80%. 85%,90%, 95%, 98% or 99% of the antibodies in said first antibodycomposition have no more or no less than (m) functionalized acceptorglutamine residues (Q) per antibody, wherein m is an integer, e.g. m=1,2 or 4. Optionally, at least 70%, 80%, 90%, 95%, 98% or 99% of theantibodies in the composition have the same q, r and z values. It can bespecified that the antibodies will share the same —NH—(C)_(n)—X, L, V,V′, Y, Y′, R, RR′ and/or Z moieties.

In one aspect the invention provides an antibody composition ofantibodies (e.g. a plurality tetrameric or full-length antibodies)linked (covalently) to a moiety of interest (Z), preferably via alinker, wherein the composition is characterized by a mean Z:antibodyratio (e.g. mean DAR) of close to 2 (e.g., between 1.5 and 2.0, orbetween 1.7 and 2.0, between 1.8 and 2.0, or between 1.9 and 2.0) lessthan 10%, less than 5%, less than 2% or less than 1% of the antibodiesin the composition comprise more than two moieties of interest (Z) perantibody. Preferably the composition is substantially free of antibodieshaving more than 2 moieties of interest per antibody. In one embodiment,the composition is a composition of antibodies of Formula IVa or IVb.

In one embodiment, the invention provides a composition of a pluralityof antibodies (e.g. a plurality tetrameric or full-length antibodies)linked (covalently) to a moiety of interest (Z), preferably via alinker, wherein the antibodies have a mean Z:antibody ratio (e.g. meanDAR) of at least 1.5, 1.6, 1.7 or 1.8, wherein less than 10%, less than5%, less than 2% or less than 1% of the antibodies in the compositioncomprise more than two moieties of interest (Z) per antibody.Preferably, less than 25%, 20%, 15% or preferably 10% of the antibodiesin the composition comprise less than two moieties of interest (Z) perantibody and/or less than 25%, 20%, 15% or preferably 10% of theantibodies comprise less than two functionalized acceptor glutamines perantibody. In one embodiment, the composition is a composition ofantibodies of Formulae II, IVa or IVb.

In one embodiment, the invention provides a composition of antibodies(e.g. a plurality tetrameric or full-length antibodies) linked(covalently) to a moiety of interest (Z), preferably via a linker,wherein:

-   -   the antibodies have a mean Z:antibody ratio (e.g. mean DAR) of        at least 1.5, 1.6, 1.7 or 1.8 (e.g. between 1.5, 1.6, 1.7 or 1.8        and 2.0),    -   less than 10%, less than 5%, or less than 2% of the antibodies        comprise more than two functionalized acceptor glutamines per        antibody, and    -   less than 25%, 20%, 15% or preferably 10% of the antibodies        comprise less than two moieties of interest (Z) per antibody        and/or less than 25%, 20%, 15% or preferably 10% of the        antibodies comprise less than two functionalized acceptor        glutamines per antibody.

Optionally, the antibodies are linked to said moiety of interest (Z) viaone functionalized acceptor amino acid (e.g. glutamine, a functionalizedacceptor glutamine of Formula II, IVa or IVb) on each heavy chain of theantibody. Optionally, at least 70%, 80%. 85%, 90%, 95%, 98% or 99% ofthe antibodies in the composition comprise one functionalized acceptoramino acid (e.g. glutamine, a functionalized acceptor glutamine ofFormula II, IVa or IVb) on each heavy chain

In one embodiment, the invention provides a composition of a pluralityof full-length antibodies comprising one acceptor glutamine in eachheavy chain, preferably wherein said antibodies share the same primaryamino acid sequence, wherein at least 70%, 80%. 85%, 90%, 95%, 98% or99% of the antibodies in the composition comprise one functionalizedacceptor amino acids (e.g. glutamine, a functionalized acceptorglutamine of Formula II, IVa or IVb) on each heavy chain

In one aspect the invention provides an antibody composition ofantibodies (e.g. a plurality of tetrameric or full-length antibodies)linked (covalently) to a moiety of interest (Z), preferably via alinker, wherein the composition is characterized by a mean Z:antibodyratio (e.g. mean DAR) of close to 4 (e.g., between 3.0 and 4.0, orbetween 3.5 and 4.0, or between 3.6 and 4.0) wherein less than 10%, lessthan 5%, or less than 2% of the antibodies comprise more than fourfunctionalized acceptor amino acids (e.g., glutamines) per antibody.Preferably, the composition is substantially free of antibodies havingmore than 4 moieties of interest (Z) per antibody. In one embodiment,the composition is a composition of antibodies of Formula IVb.

In one embodiment, the invention provides a composition of antibodies(e.g. a plurality of tetrameric or full-length antibodies) covalentlylinked to a moiety of interest (Z), preferably via a linker, wherein theantibodies have a mean Z:antibody ratio (e.g. mean DAR) of at least 3.2,3.4, 3.5 or 3.6, wherein less than 10%, less than 5%, or less than 2% ofthe antibodies comprise more than four functionalized acceptor aminoacids (e.g. glutamines) per antibody. In one embodiment, the compositionis a composition of antibodies of Formula IVb.

Optionally, the antibodies are linked to a moiety of interest (Z) oneach of two functionalized acceptor amino acids (e.g. glutamine, afunctionalized acceptor glutamine of Formula II, IVa or IVb) on eachheavy chain of the antibody. Optionally, at least 70%, 80%, 85%, 90% ofthe antibodies in the composition comprise two functionalized acceptorglutamines (e.g. a functionalized acceptor glutamine of Formula II orIVb) on each heavy chain

In one embodiment, the invention provides a composition of a pluralityof (e.g. tetrameric or full-length) antibodies comprising one acceptoramino acid (e.g. glutamine) in each heavy chain, preferably wherein saidantibodies share the same primary amino acid sequence, wherein at least70%, 80%, 85%, 90% of the antibodies in the composition comprise twofunctionalized acceptor glutamines (e.g. a functionalized acceptorglutamine of Formula II or IVb) on each heavy chain

In one embodiment, the invention provides a composition comprising aplurality of antibodies comprising one acceptor glutamine on each heavychain, wherein at least 70%, 80%, 85% or 90%, of the antibodies in thecomposition comprise on each heavy chain one functionalized acceptorglutamine residue (Q) having Formula IVa, below,

(Q)-NH—(C)_(n)—X-L-(V—(Y—(Z)_(z))_(q))_(r)  Formula IVa

Preferably the compositions are substantially free of antibodies havingmore than 2 moieties of interest (Z) and/or antibodies having more onefunctionalized acceptor glutamine per antibody.

In one embodiment, the invention provides a composition comprising aplurality of antibodies comprising two acceptor glutamines on each heavychain, wherein at least 70%, 80%, 85% or 90%, of the antibodies in thecomposition comprise on each heavy chain two functionalized acceptorglutamine residue (Q) having Formula IVb, below,

(Q)-NH—(C)_(n)—X-L-(V—(Y-(M)_(z))_(q))_(r)  Formula IVb.

Preferably the compositions are substantially free of antibodies havingmore than 4 moieties of interest (Z) and/or antibodies having more onefunctionalized acceptor glutamine per antibody.

In one embodiment of any of the compositions of the invention,substantially all of the antibodies in the composition share the sameprimary amino acid sequence. In one embodiment of any of thecompositions Z is optionally a hydrophobic compound. In one embodimentof any of the compositions Z is optionally an organic compound having amolecular weight of at least 400 g/mol, 500 g/mol, 600 g/mol, 700 g/mol,800 g/mol, 900 g/mol or 1000 g/mol. In one embodiment of any of thecompositions Z is optionally a hydrophobic compound. In one embodimentof any of the compositions Z is optionally a negatively chargedcompound. In one embodiment of any of the compositions, the moiety ofinterest (Z) is optionally selected from the group consisting oftaxanes, anthracyclines, camptothecins, epothilones, mytomycins,combretastatins, vinca alkaloids, nitrogen mustards, maytansinoids,calicheamycins, duocarmycins, tubulysmes, dolastatins and auristatins,enediynes, pyrrolobenzodiazepines, and ethylenimines.

According to another embodiment, the invention enables two or moreantibody compositions to be prepared that share the same antibody moietyand the same antibody:conjugate stoichiometry, e.g., compositions may becharacterized by any of the properties of antibody compositions above,or otherwise described herein. Such compositions advantageously providethe possibility to compare two compositions that differ in a selectedcomponent of the lysine-based linker, e.g., the structure or number ofV, V′, Y, Y′, R, RR′, and/or Z moieties (including the presence orabsence thereof), or that differ in the antibody component (e.g. thesequence of a heavy and/or light chain) including antibodies that bindto the same predetermined antigen(s) or to different predeterminedantigen(s) (e.g. for evaluating drug targets). In one embodiment,provided is a first and a second antibody composition, preferably inseparate containers, where in each of said first and second compositionscomprise a plurality of antibodies of Formula II, IVa or IVb, wherein atleast 70%, 80%, 90%, 95%, 98% or 99% of the antibodies in thecomposition have the same number of functionalized acceptor glutamineresidues (Q) (e.g., a functionalized acceptor glutamine of Formula II,IVa or IVb) per antibody, further wherein said number, m, offunctionalized acceptor glutamine residues is substantially the same forsaid first and second composition. Preferably, the number is 1, 2 or 4.Optionally, said first and second composition differ from one another inone, two, three or more of elements V, V′, Y, Y′, R, RR′ and/or Z.

In another embodiment, the invention comprises a method (e.g. a methodof designing, evaluating, comparing, optimizing, testing or making anantibody), the method comprising:

(a) providing a first antibody composition comprising a plurality ofantibodies of Formula II, IVa or IVb, wherein at least 70%, 80%, 90%,95%, 98% or 99% of the antibodies in the composition have the samenumber of functionalized acceptor glutamine residues (Q) per antibody;

(b) providing a second antibody composition comprising a plurality ofantibodies of Formula II, IVa or IVb, wherein at least 70%, 80%, 90%,95%, 98% or 99% of the antibodies in the composition have the samenumber of functionalized acceptor glutamine residues (Q) per antibody,optionally wherein the number of functionalized acceptor glutamineresidues is substantially the same for said first and secondcomposition, optionally, the number is 1, 2 or 4; and

(c) evaluating said first and second composition, optionally comparingsaid first composition to said second composition.

The functionalized acceptor glutamine residues are preferably afunctionalized acceptor glutamine of Formula II, IVa or IVb). The step(c) of evaluating or comparing may comprise evaluating a composition forany desired characteristic, e.g. for biological activity, any physicalor pharmacokinetic properties. The composition of (a) and (b) arepreferably provided in separate containers. Optionally, said first andsecond composition differ from one another in one, two, three or more ofelements V, V′, Y, Y′, R, RR′ and/or Z; in one embodiment, the chemicalstructure of one or more of elements V, Y, Z, R, R′ and/or Z differ; inone embodiment, one or more of elements V, V′, Y, Y′, R, RR′ and/or Zare absent in the first or second composition but present in the secondcomposition.

In one embodiment, each of the first and second antibody compositions ofsteps (a) and (b) are obtained by a method comprising: (i) providing anantibody composition of Formula II; and (ii) reacting said antibodycomposition of Formula II with a compound of Formula III. Preferably thecompound of Formula III in steps (a)(ii) differs from compound ofFormula III in step (b)(ii) in one, two, three or more of elements V′,Y′, Z, R′ and/or Z; in one embodiment, the chemical structure of one ormore of elements V′, Y′, Z, R′ and/or Z differ; in one embodiment, oneor more of elements L, V, V′, Y, Y′, Z, R, RR′ and/or Z are absent inthe first or second composition but present in the second composition.Optionally, the reaction conditions of step (a)(ii) and (b)(ii) aresubstantially the same.

In one embodiment, the invention provides a kit comprising at least two(e.g. at least 2, 3, 4, 5, etc.) antibody compositions comprising aplurality of antibodies of Formula II, IVa or IVb, wherein at least 70%,80%, 90%, 95%, 98% or 99% of the antibodies in the composition have thesame number of functionalized acceptor glutamine residues (Q) perantibody, wherein the kit comprises a first antibody composition and asecond antibody composition in separate containers, and wherein thesecond antibody composition comprises antibodies having at least one X,L, V, Y, L′, V′, Y′, (RR′) and/or Z moiety that differs from arespective X, L, V, Y, L′, V′, Y′, (RR′) and/or Z moiety of said firstantibody. Optionally, the antibodies of the first and second antibodycomposition share the same heavy and/or light chain amino acidsequences. In one embodiment, the invention provides a kit comprising atleast two antibody compositions comprising a plurality of antibodies ofFormula II, IVa or IVb, wherein at least 70%, 80%, 90%, 95%, 98% or 99%of the antibodies in the composition have the same number offunctionalized acceptor glutamine residues (Q) per antibody, wherein thekit comprises a first antibody composition and a second antibodycomposition in separate containers, and wherein the second antibodycomposition comprises antibodies differ in their heavy and/or lightchain amino acid sequences. In one embodiment, the antibodies of thefirst and second antibody compositions are directed to the samepredetermined antigen. In one embodiment, the antibodies of the firstand second antibody compositions are directed to a differentpredetermined antigen and differ in their heavy and/or light chain aminoacid sequences.

According to one embodiment, the acceptor glutamine residue that isfunctionalized, e.g., according to Formula II, IVa or IVb, is part ofthe primary structure of the antibody. Preferably, the functionalizedacceptor glutamine residue (Q) is part of the primary structure of aheavy chain. Optionally, the antibody comprises two heavy chains, eachof which comprise a functionalized acceptor glutamine residue (Q) ofFormula II, IVa or IVb.

In one embodiment, the antibody of the invention comprises a constantregion and/or Fc region of human origin, optionally a human IgG1, IgG2,IgG3 or IgG4 isotype. In one embodiment, the antibody comprises awild-type (naturally occurring) human heavy and/or light chain constantregion sequence (representing a full-length human constant region or afragment thereof, e.g. a contiguous sequence of at least 20, 50, 60, 75or 100 amino acid residues of a human constant region). Preferably theantibody comprises a human heavy and/or light chain constant region(e.g. a full-length heavy and/or light chain human constant region) thatis at least 95, 98, or 99% identical to a naturally occurring humanconstant region sequence. Optionally, the constant region furthercomprises one or more (e.g. 2, 3, 4, 5 or more) amino acidsubstitution(s), optionally wherein said substitution(s) is thereplacement of an amino acid residue by a glutamine residue, optionallywherein said substitution(s) is the replacement of an amino acid residueby a non-glutamine, non-asparagine and/or non-aspartic acid, or by anon-negatively charged residue. Optionally, the wild-type constantregion sequence comprises one or more single amino acid substitutions.Optionally, the wild-type constant region sequence comprises one or moreamino acid substitutions, wherein all the substitutions are naturallyoccurring amino acids. Preferably the constant region sequence is freeof an introduced enzymatic recognition tag, i.e. a sequence of 2, 3, 4,5 or more residues not naturally present in the constant region sequenceand specifically recognized by an enzyme, for example an enzyme thatconjugates a moiety of interest to an antibody, aformylglycine-generating enzyme, a sortase, etc.

In one embodiment, the antibody (i.e. the “Ab”) is an antibodycomprising a Fc domain or portion thereof comprising an acceptorglutamine residue. In one embodiment, the antibody is an antibodyfragment, e.g. a single chain antibody, comprising an acceptor glutamineresidue, optionally wherein the antibody fragment comprises a peptide“tag” comprising an acceptor glutamine residue.

In one embodiment, an antibody comprises a functionalized acceptorglutamine residue (Q) at position 295 of a heavy chain of an antibody.

In one embodiment of any of the methods or antibodies of the invention,an acceptor glutamine in an antibody or antibody fragment is flanked atthe +2 position (e.g. residue 297 of the heavy chain, according to theEU index numbering system) by a non-aspartic acid residue. In oneembodiment, the residue at the +2 position is a non-aspartic acid,non-glutamine residue. In one embodiment, the residue at the +2 positionis a non-aspartic acid, non-asparagine residue. In one embodiment, theresidue at the +2 position is a non-negatively charged amino acid.

In one embodiment, the antibody is a modified antibody comprising anintroduced acceptor glutamine residue. In one embodiment, an antibodycomprises a functionalized acceptor glutamine residue (Q) at position297 of a heavy chain of an antibody. In one embodiment, an antibodycomprises a functionalized acceptor glutamine residue (Q) at position295 and 297 of a heavy chain of an antibody.

In one embodiment, an antibody comprises a functionalized acceptorglutamine residue (Q) at position 295 and lacks an acceptor glutamine atposition 297 of a heavy chain of an antibody.

In one embodiment, an antibody comprises a functionalized acceptorglutamine residue (Q) at position 297 and lacks an acceptor glutamineposition 295 of a heavy chain of an antibody.

In one embodiment, an antibody is capable of being internalized intocells that express an antigen to which the antibody binds (e.g. a tumoror viral antigen) and/or induces internalization of the antigen on saidantigen-expressing cells.

In one embodiment, the invention provides an antibody or antibodyfragment comprising a functionalized acceptor glutamine residue, thefunctionalized acceptor glutamine residue having Formula IVa,

(Q)-NH—(C)_(n)—X-L-(V—(Y—(Z)_(z))_(q))_(r)  Formula IVa

or a pharmaceutically acceptable salt or solvate thereof,

wherein:

Q is a glutamine residue present in an antibody or antibody fragment;

(C)_(n) is a substituted or unsubstituted alkyl or heteroalkyl chain,optionally wherein any carbon of the chain is substituted with analkoxy, hydroxyl, alkylcarbonyloxy, alkyl-S—, thiol, alkyl-C(O)S—,amine, alkylamine, amide, or alkylamide;

n is an integer selected from among the range of 2 to 20;

X is NH, O, S, absent, or a bond;

L is independently absent, a bond or a continuation of a bond, or acarbon comprising framework of 5 to 200 atoms substituted at one or moreatoms;

r is an integer selected from among 1, 2, 3 or 4;

q is an integer selected from among 1, 2, 3 or 4;

z is an integer selected from among 1, 2, 3 or 4; and

V is independently absent, a bond or a continuation of a bond, anon-cleavable moiety or a conditionally-cleavable moiety;

Y is independently absent, a bond or a continuation of a bond, or aspacer system which is comprised of 1 or more spacers; and

Z is a moiety that improves pharmacokinetic properties, a therapeuticmoiety or a diagnostic moiety, wherein Z is an organic compound that iselectrically negatively charged, hydrophobic and/or that has a molecularweight of at least 400 g/mol. In one embodiment, said acceptor glutamineresidue is flanked at position +2 by a non-aspartic acid residue. In oneembodiment, said acceptor glutamine residue is flanked at position +2 bya non-aspartic acid, non-glutamine residue.

In one embodiment, the invention provides an antibody or antibodyfragment comprising an acceptor glutamine residue flanked at the +2position by a non-aspartic acid residue, wherein the acceptor glutamineresidue is functionalized with a compound comprising amoiety-of-interest. In one embodiment, said moiety-of-interest iscovalently bound to the acceptor glutamine residue via a linkercomprising a NH—(C)_(n) group, wherein (C)_(n) is a substituted orunsubstituted alkyl or heteroalkyl chain, wherein any carbon of thechain is optionally substituted with an alkoxy, hydroxyl,alkylcarbonyloxy, alkyl-S—, thiol, alkyl-C(O)S—, amine, alkylamine,amide, or alkylamide; and n is an integer selected from among the rangeof 2 to 20. In one embodiment, the functionalized acceptor glutamineresidue has a structure of Formula IVa.

In one embodiment, the invention provides an antibody or antibodyfragment comprising a functionalized acceptor glutamine residue, thefunctionalized acceptor glutamine residue having Formula IVa,

(Q)-NH—(C)_(n)—X-L-(V—(Y—(Z)_(z))_(q))_(r)  Formula IVa

or a pharmaceutically acceptable salt or solvate thereof,

wherein:

Q is a glutamine residue present in an antibody or antibody fragment;

(C)_(n) is a substituted or unsubstituted alkyl or heteroalkyl chain,optionally wherein any carbon of the chain is substituted with analkoxy, hydroxyl, alkylcarbonyloxy, alkyl-S—, thiol, alkyl-C(O)S—,amine, alkylamine, amide, or alkylamide;

n is an integer selected from among the range of 2 to 20;

X is NH, O, S, absent, or a bond;

L is independently absent, a bond or a continuation of a bond, or acarbon comprising framework of 5 to 200 atoms substituted at one or moreatoms;

r is an integer selected from among 1, 2, 3 or 4;

q is an integer selected from among 1, 2, 3 or 4;

z is an integer selected from among 1, 2, 3 or 4; and

V is independently absent, a bond or a continuation of a bond, anon-cleavable moiety or a conditionally-cleavable moiety;

Y is independently absent, a bond or a continuation of a bond, or aspacer system which is comprised of 1 or more spacers; and

Z is a a cytotoxic anti-cancer agent, optionally wherein the agentcomprises a polycyclic or macrocyclic group, optionally wherein theagent furthermore has a molecular weight of at least 400 g/mol. In oneembodiment, said acceptor glutamine residue is flanked at position +2 bya non-aspartic acid residue. In one embodiment, said acceptor glutamineresidue is flanked at position +2 by a non-aspartic acid, non-glutamineresidue.

In one embodiment, the invention provides an antibody or antibodyfragment comprising a functionalized acceptor glutamine residue, whereinsaid acceptor glutamine residue is flanked at the +2 position by anon-aspartic acid, non glutamine, residue, the functionalized acceptorglutamine residue having Formula IVa,

(Q)-NH—(C)_(n)—X-L-(V—(Y—(Z)_(z))_(q))_(r)  Formula IVa

or a pharmaceutically acceptable salt or solvate thereof,

wherein:

Q is a glutamine residue present in an antibody or antibody fragment;

(C)_(n) is a substituted or unsubstituted alkyl or heteroalkyl chain,optionally wherein any carbon of the chain is substituted with analkoxy, hydroxyl, alkylcarbonyloxy, alkyl-S—, thiol, alkyl-C(O)S—,amine, alkylamine, amide, or alkylamide;

n is an integer selected from among the range of 2 to 20;

X is NH, O, S, absent, or a bond;

L is independently absent, a bond or a continuation of a bond, or acarbon comprising framework of 5 to 200 atoms substituted at one or moreatoms, optionally, wherein the carbon comprising framework comprises alinear framework of 5 to 30 carbon atoms optionally substituted at oneor more atoms, optionally wherein the carbon comprising framework is alinear hydrocarbon, a symmetrically or asymmetrically branchedhydrocarbon, monosaccharide, disaccharide, linear or branchedoligosaccharide (asymmetrically branched or symmetrically branched),other natural linear or branched oligomers (asymmetrically branched orsymmetrically branched), or a dimer, trimer, or higher oligomer (linear,asymmetrically branched or symmetrically branched) resulting from anychain-growth or step-growth polymerization process;

r is an integer selected from among 1, 2, 3 or 4;

q is an integer selected from among 1, 2, 3 or 4;

z is an integer selected from among 1, 2, 3 or 4; and

V is independently absent, a non-cleavable moiety or aconditionally-cleavable moiety;

Y is independently absent, a bond or a continuation of a bond, or aspacer system which is comprised of 1 or more spacers; and

Z is a moiety that improves the pharmacokinetic properties, atherapeutic moiety or a diagnostic moiety. In one embodiment, Z is anorganic compound that is charged, hydrophobic and/or has a molecularweight of at least 400 g/mol. In one embodiment, the antibody comprisesone acceptor glutamine on each heavy chain

In one embodiment, the invention provides antibody or antibody fragmentcomprising a functionalized acceptor glutamine residue, wherein saidacceptor glutamine residue is flanked at the +2 position by an aminoacid other than aspartic acid or glutamine, the functionalized acceptorglutamine residue having Formula IVa,

(Q)-NH—(C)_(n)—X-L-(V—(Y—(Z)_(z))_(q))_(r)  Formula IVa

or a pharmaceutically acceptable salt or solvate thereof,

wherein:

Q is a glutamine residue present in an antibody or antibody fragment;

(C)_(n) is a substituted or unsubstituted alkyl or heteroalkyl chain,optionally wherein any carbon of the chain is substituted with analkoxy, hydroxyl, alkylcarbonyloxy, alkyl-S—, thiol, alkyl-C(O)S—,amine, alkylamine, amide, or alkylamide;

n is an integer selected from among the range of 2 to 20;

X is NH, O, S, absent, or a bond;

L is independently absent, a bond or a continuation of a bond, or acarbon comprising framework of 5 to 200 atoms substituted at one or moreatoms, optionally, wherein the carbon comprising framework comprises alinear framework of 5 to 30 carbon atoms optionally substituted at oneor more atoms, optionally wherein the carbon comprising framework is alinear hydrocarbon, a symmetrically or asymmetrically branchedhydrocarbon, monosaccharide, disaccharide, linear or branchedoligosaccharide (asymmetrically branched or symmetrically branched),other natural linear or branched oligomers (asymmetrically branched orsymmetrically branched), or a dimer, trimer, or higher oligomer (linear,asymmetrically branched or symmetrically branched) resulting from anychain-growth or step-growth polymerization process;

r is an integer selected from among 1, 2, 3 or 4;

q is an integer selected from among 1, 2, 3 or 4;

z is an integer selected from among 1, 2, 3 or 4; and

V is independently absent, a non-cleavable moiety or aconditionally-cleavable moiety;

Y is independently absent, a bond or a continuation of a bond, or aspacer system which is comprised of 1 or more spacers; and

Z is a moiety of interest.

In one embodiment, the invention provides an antibody or antibodyfragment comprising a functionalized acceptor glutamine residue havingFormula II:

(Q)-NH—(C)_(n)—X-L-(V—(Y—(R)_(z))_(q))_(r)  Formula II

or a pharmaceutically acceptable salt or solvate thereof,

wherein:

Q is a glutamine residue present in an antibody or antibody fragment;

(C)_(n) is a substituted or unsubstituted alkyl or heteroalkyl chain,optionally wherein any carbon of the chain is optionally substitutedwith alkoxy, hydroxyl, alkylcarbonyloxy, alkyl-S—, thiol, alkyl-C(O)S—,amine, alkylamine, amide, or alkylamide;

n is an integer from among the range of 2 to 20;

X is NH, O, S, absent, or a bond;

L is independently absent, a bond or a continuation of a bond, or acarbon comprising framework of 1 to 200 atoms substituted at one or moreatoms, optionally wherein the carbon comprising framework comprises alinear framework of 3 to 30 carbon atoms optionally substituted at oneor more atoms, optionally wherein the carbon comprising framework is alinear hydrocarbon, a symmetrically or asymmetrically branchedhydrocarbon, monosaccharide, disaccharide, linear or branchedoligosaccharide (asymmetrically branched or symmetrically branched),other natural linear or branched oligomers (asymmetrically branched orsymmetrically branched), or a dimer, trimer, or higher oligomer (linear,asymmetrically branched or symmetrically branched) resulting from anychain-growth or step-growth polymerization process;

r is an integer selected from among 1, 2, 3 or 4;

q is an integer selected from among 1, 2, 3 or 4;

z is an integer selected from among 1, 2, 3 or 4; and

V is independently absent, a bond or a continuation of a bond, anon-cleavable moiety or a conditionally-cleavable moiety;

Y is independently absent, a bond or a continuation of a bond, or aspacer system which is comprised of 1 or more spacers; and

R is a reactive moiety.

In one embodiment, the invention provides an An antibody or antibodyfragment comprising a functionalized acceptor glutamine residue havingFormula IVb,

(Q)-NH—(C)—X-L-(V—(Y-(M)_(z))_(q))_(r)  Formula IVb

or a pharmaceutically acceptable salt or solvate thereof,

wherein:

Q is a glutamine residue present in an antibody or antibody fragment;

(C)_(n) is a substituted or unsubstituted alkyl or heteroalkyl chain,optionally wherein any carbon of the chain is substituted with a alkoxy,hydroxyl, alkylcarbonyloxy, alkyl-S—, thiol, alkyl-C(O)S—, amine,alkylamine, amide, or alkylamide;

n is an integer selected from among the range of 2 to 20;

X is NH, O, S, absent, or a bond;

L is independently absent, a bond or a continuation of a bond, or acarbon comprising framework of 1 to 200 atoms substituted at one or moreatoms, optionally, wherein the carbon comprising framework comprises alinear framework of 3 to 30 carbon atoms optionally substituted at oneor more atoms, optionally wherein the carbon comprising framework is alinear hydrocarbon, a symmetrically or asymmetrically branchedhydrocarbon, monosaccharide, disaccharide, linear or branchedoligosaccharide (asymmetrically branched or symmetrically branched),other natural linear or branched oligomers (asymmetrically branched orsymmetrically branched), or a dimer, timer, or higher oligomer (linear,asymmetrically branched or symmetrically branched) resulting from anychain-growth or step-growth polymerization process;

r is an integer selected from among 1, 2, 3 or 4;

q is an integer selected from among 1, 2, 3 or 4;

z is an integer selected from among 1, 2, 3 or 4;

V is independently absent, a bond or a continuation of a bond, anon-cleavable moiety or a conditionally-cleavable moiety;

Y is independently absent, a bond or a continuation of a bond, or aspacer system which is comprised of 1 or more spacers;

M is independently: R or (RR′)-L′-(V′—(Y′—(Z)_(z′))_(q′))_(r′), wherein

R is a reactive moiety of Formula II;

(RR′) is an addition product between Rand a complementary reactivemoiety R′;

L′ is independently absent, a bond or a continuation of a bond, or acarbon comprising framework of 1 to 200 atoms substituted at one or moreatoms, optionally, wherein the carbon comprising framework comprises alinear framework of 3 to 30 carbon atoms optionally substituted at oneor more atoms, optionally wherein the carbon comprising framework is alinear hydrocarbon, a symmetrically or asymmetrically branchedhydrocarbon, monosaccharide, disaccharide, linear or branchedoligosaccharide (asymmetrically branched or symmetrically branched),other natural linear or branched oligomers (asymmetrically branched orsymmetrically branched), or a dimer, trimer, or higher oligomer (linear,asymmetrically branched or symmetrically branched) resulting from anychain-growth or step-growth polymerization process;

V′ is independently absent, a bond or a continuation of a bond, anon-cleavable moiety or a conditionally-cleavable moiety;

Y′ is independently absent, a bond or a continuation of a bond, or aspacer system which is comprised of 1 or more spacers;

Z is independently a reactive group, a moiety that improves thepharmacokinetic properties, a therapeutic or diagnostic moiety, and eachZ is directly coupled to either Y or V when Y is absent, or L when bothY and V are absent; and

z′, q′ and r′ are each independently an integer selected from among 1,2, 3 or 4. In one embodiment, RR′ is a thio-maleimide (orhalo-acetamide) addition, for example, aN,S-disubstituted-3-thio-pyrrolidine-2,5-dione; Staudinger ligation, forexample, a N,3- or N,4-substitued-5-dipenylphosphinoxide-benzoic amide;Huisgen 1,3-cycloaddition (click reaction), for example, a N,S-disubstituted-3-thio-pyrrolidine-2,5-dione,1,4-disubstituted-1,2,3-triazole, 3,5-disubstituted-isooxazole, or3,5-disubstituted-tetrazole; Diels-Alder cycloaddition adduct, forexample the 2,4-cycloaddition product between an O orN-substituted-5-norbornene-2-carboxylic ester or amide,N-substituted-5-norbornene-2,3-dicarboxylic imide, O orN-substituted-7-oxonorbornene-5-carboxylic ester or amide, orN-substituted-7-oxonorbornene-5,6-dicarboxylic imide and a 9-substitutedanthracene or 3-substituted 1,2,4,5-tetrazine; or any high yieldselective amidation or imidization reaction.

In one embodiment of Formulae II or IVb, said acceptor glutamine residueis flanked at the +2 position by a non-aspartic acid residue. In oneembodiment, said amino acid residue at the +2 position is not aglutamine. In one embodiment, said functionalized acceptor glutamineresidue is in an antibody heavy chain, optionally within the CH2 domain.In one embodiment, said functionalized acceptor glutamine residue is inan antibody heavy chain at position 295 (EU numbering). In oneembodiment, said functionalized acceptor glutamine residue is in anantibody heavy chain at position 297 (EU numbering). In one embodiment,said antibody comprises a N297X or Q295X (e.g., Q295X/N297Q)substitution, wherein X is any amino acid other than aspartic acid. Inone embodiment, said antibody comprises a N297X or Q295X (e.g.,Q295X/N297Q) substitution, wherein X is any amino acid other thanaspartic acid, asparagine or glutamine. In one embodiment, said antibodycomprises an asparagine at residue 297 that substantially lacks N-linkedglycosylation. In one embodiment, said antibody comprises a T299Xsubstitution, wherein X is any amino acid other than threonine andresults in lack of N-linked glycosylation at amino acid residue N297. Inone embodiment, said antibody is produced in a host cell that producesantibodies lacking N-linked glycosylation at amino acid residue N297.

In one embodiment of Formulae II or IVb, R or R′ is a moiety comprisinga bioorthogonal-reaction compatible reactive group, for example anunprotected or protected thiol, epoxide, maleimide, haloacetamide,o-phoshenearomatic ester, azide, fulminate, sulfonate ester, alkyne,cyanide, amino-thiol, carbonyl, aldehyde, generally any group capable ofoxime and hydrazine formation, 1,2,4,5-tetrazine, norbornene, otherstained or otherwise electronically activated alkene, a substituted orunsubstituted cycloalkyne, generally any reactive groups which form viabioorthogonal cycloaddition reaction a 1,3- or 1,5-disubstitutedtriazole, any diene or strained alkene dienophile that can react viainverse electron demand Diels-Alder reaction, a protected or unprotectedamine, a carboxylic acid, an aldehyde, an oxyamine

In one embodiment of Formulae II or IVb, (C)_(n), L and/or R do notcomprise a cyclic group. In one embodiment of Formulae II or IVb, n isan integer from among the range of 2 to 10, L is absent, and wherein Rdoes not comprise a cyclic group. In one embodiment, R is an azide. Inone embodiment of Formulae II or IVb, R′ comprises a cyclic group. Inone embodiment of Formulae II or IVb, n is an integer from among therange of 10 to 20, and R comprises a cyclic group. In one embodiment ofFormulae II or IVb, L is present and R comprises a cyclic group. In oneembodiment R′ is an azide. In any embodiment said cyclic group may be apolycyclic group; optionally, the cyclic group is a cyclooctyne,optionally a substituted or unsubstituted dibenzylcycolooctyne.

In one embodiment of Formulae II or IVb, V is absent and V′ is present,or V′ is absent and V is present. In one embodiment of Formulae II orIVb, Y is absent and Y′ is present, or Y′ is absent and Y is present.

In any embodiment of Formulae I to IV, n may be an integer from amongthe range of 10 to 20. In any embodiment of Formulae I to IV, (C)_(n) isa heteroalkyl chain comprising a (CH₂—CH₂—O—)_(x) group, wherein x is aninteger from among the range of 1 to 6. In any embodiment of Formulae Ito IV, at least one of L, V or Y can be specified as being present. Inany embodiment of Formulae I to IV, n may be an integer from among therange of 2 to 6 and at least one of L, V or Y are present.

In one embodiment of Formulae I to IV, (C)_(n) comprises an amino acidor a di-, tri-, tetra, or oligopeptide. In one embodiment of Formulae Ito IV, NH—(C)_(n) comprises NH—O—(CH₂)_(n)—. In one embodiment ofFormulae I to IV, (C)_(n) is a substituted or unsubstituted alkyl orheteroalkyl chain, wherein the carbon adjacent to the nitrogen isunsubstituted. In one embodiment of Formulae I to IV (C)_(n), is asubstituted or unsubstituted carbon chain, wherein the carbon adjacentto the nitrogen is unsubstituted and wherein any carbon of the chainother than the carbon adjacent to the nitrogen is optionally substitutedwith a O, N or S atom of an ether, ester, thioether, thioester, amine,alkylamine, amide, or alkylamide; n, the length of the carbon chain, is2 to 20 atoms. In one embodiment of Formulae I to IV, L comprises alinear carbon comprising framework of 5 to 30 carbon atoms optionallysubstituted at one or more atoms. In one embodiment of Formulae I to IV,L comprises a (CH₂—CH₂—O—)_(x) group, wherein x is an integer from amongthe range of 1 to 10. In one embodiment of Formulae I to IV, the groups—(C)_(n)—X-L- collectively comprise a structure CH₂—(CH₂—O—CH₂)_(n)—CH₂or (CH₂—CH₂—O—)_(n), wherein x is an integer from among the range of 3to 24. In one embodiment of Formulae I to IV L comprises an amino acidor a di-, tri-, tetra-, or oligopeptide. In one embodiment of Formulae Ito IV L is a carbon framework of:

a) 2-15 linear carbon atoms optionally substituted at one or more atoms;

b) 3-15 linear carbon atoms optionally substituted at one or more atoms;

c) 5-15 linear carbon atoms optionally substituted at one or more atoms;

d) 5-20 linear carbon atoms optionally substituted at one or more atoms;

e) 3-30 linear carbon atoms optionally substituted at one or more atoms;

f) 5-30 linear carbon atoms optionally substituted at one or more atoms;or

g) 3, 4, 5 or 6 linear carbon atoms optionally substituted at one ormore atoms.

In one embodiment, said carbon-comprising framework of linear carbonatoms is unsubstituted.

In one embodiment of Formulae I to IV, V comprises a di-, tri-, tetra-,or oligopeptide. In one embodiment, V is a conditionally-cleavablemoiety following prior conditional transformation, which can be cleavedor transformed by a chemical, photochemical, physical, biological, orenzymatic process. In one embodiment, V comprises a (CH₂—CH₂—O—)_(x)group, wherein x is an integer from among the range of 1 to 10.

In one embodiment of Formulae I to IV, Y is a self-eliminating spacersystem. In one embodiment Y is a non-self-elimination spacer system.

In one embodiment of Formulae I to IV, any of r, r′, q and/or q′represent the degree of branching. In one embodiment of Formulae I toIV, any of r, r′, q and/or q′ represent the degree of polymerization.

In one embodiment, the invention provides a composition comprising aplurality of antibodies of Formulae IVa sharing the same heavy and/orlight chain amino acid sequence, wherein at least 90% of the antibodiesin said composition have (m) functionalized acceptor glutamine residues(Q) per antibody. In one embodiment m=2. In one embodiment, theinvention provides a composition comprising a plurality of antibodies ofFormulae II or IV sharing the same heavy and/or light chain amino acidsequence, wherein at least 90% of the antibodies in said compositionhave at least (m) functionalized acceptor glutamine residues (Q) perantibody. In one embodiment m=4.

In one embodiment, the invention provides a composition comprising aplurality of antibodies comprising one acceptor glutamine on each heavychain, wherein at least 80% of the antibodies in the compositioncomprise on each heavy chain one functionalized acceptor glutamineresidue (Q) of any one of Formulae II-IV. In one embodiment, theinvention provides a composition comprising a plurality of antibodiescomprising one acceptor glutamine on each heavy chain, wherein at least80% of the antibodies in the composition comprise on each heavy chaintwo functionalized acceptor glutamine residues (Q) of any one ofFormulae II-IV.

In one embodiment, the invention provides a composition comprising aplurality of antibodies linked to a moiety of interest (Z) via onefunctionalized acceptor glutamine on each heavy chain of the antibody,wherein the composition is characterized by a mean Z:antibody ratio ofat least 1.5, 1.6, 1.7 or 1.8, wherein less than 10%, less than 5%, orless than 2% of the antibodies comprise more than two functionalizedacceptor glutamines per antibody. In one embodiment, less than 25%, 20%,15% or preferably 10% comprise less than two moieties of interest (Z)per antibody. In one embodiment, at least 80% of the antibodies in thecomposition comprise on each heavy chain one functionalized acceptorglutamine residue (Q) of any one of Formulae I-IV.

In one embodiment, the invention provides a composition comprising aplurality of antibodies linked to a moiety of interest (Z) via twofunctionalized acceptor glutamines on each heavy chain of the antibody,wherein the composition is characterized by a mean Z:antibody ratio ofat least 3.2, 3.4, 3.5 or 3.6, wherein less than 10%, less than 5%, orless than 2% of the antibodies comprise more than four functionalizedacceptor glutamines per antibody. In one embodiment, at least 80% of theantibodies in the composition comprise on each heavy chain twofunctionalized acceptor glutamine residues (Q) of any one of Formulae Ito IV.

In one embodiment, the invention provides a linking reagent, apharmaceutically acceptable salt or solvate thereof, or aprotein-conjugated linking reagent having the general Formula Ib:

G-NH—(C)_(n)—X-L-(V—(Y—(R)_(z))_(q))_(r)  Formula Ib

or a pharmaceutically acceptable salt or solvate thereof,

wherein:

G is a H, amine protecting group, or upon conjugation, an antibody orantibody fragment attached via an amide bond;

(C)_(n) is a substituted or unsubstituted alkyl or heteroalkyl chain,optionally wherein any carbon of the chain is substituted with analkoxy, hydroxyl, alkylcarbonyloxy, alkyl-S—, thiol, alkyl-C(O)S—,amine, alkylamine, amide, or alkylamide;

N is an integer selected from among the range of 2 to 20, preferably 3to 6;

X is NH, O, S, absent, or a bond;

L is independently absent, a bond or a continuation of a bond, or acarbon comprising framework of 1 to 200 atoms substituted at one or moreatoms, optionally, wherein the carbon comprising framework comprises alinear framework of 5 to 30 atoms optionally substituted at one or moreatoms, optionally wherein the carbon comprising framework is a linearhydrocarbon, a symmetrically or asymmetrically branched hydrocarbon,monosaccharide, disaccharide, linear or branched oligosaccharide(asymmetrically branched or symmetrically branched), other naturallinear or branched oligomers (asymmetrically branched or symmetricallybranched), or a dimer, trimer, or higher oligomer (linear,asymmetrically branched or symmetrically branched) resulting from anychain-growth or step-growth polymerization process;

r is an integer selected from among 1, 2, 3 or 4;

q is an integer selected from among 1, 2, 3 or 4;

z is an integer selected from among 1, 2, 3 or 4;

V is independently absent, a non-cleavable moiety or aconditionally-cleavable moiety, optionally following prior conditionaltransformation, which can be cleaved or transformed by a chemical,photochemical, physical, biological, or enzymatic process;

Y is independently absent, a bond or a continuation of a bond, or aspacer system which is comprised of 1 or more spacers; and

R is a reactive moiety.

In one embodiment, the invention provides a compound having thestructure of Formula III, below,

R′-L-(V—(Y-(M)_(z))_(q))_(r)  Formula III

or a pharmaceutically acceptable salt or solvate thereof,

wherein:

R′ is a reactive group, e.g. a reactive group capable of forming a bondwith reactive group R of Formula Ib or Formula II;

L is independently absent, or a carbon comprising framework of 1 to 200atoms substituted at one or more atoms, optionally, wherein the carboncomprising framework comprises a linear framework of 5 to 30 atomsoptionally substituted at one or more atoms, optionally wherein thecarbon comprising framework is a linear hydrocarbon, a symmetrically orasymmetrically branched hydrocarbon, monosaccharide, disaccharide,linear or branched oligosaccharide (asymmetrically branched orsymmetrically branched), other natural linear or branched oligomers(asymmetrically branched or symmetrically branched), or a dimer, timer,or higher oligomer (linear, asymmetrically branched or symmetricallybranched) resulting from any chain-growth or step-growth polymerizationprocess;

V is independently absent, a bond or a continuation of a bond, anon-cleavable moiety or a conditionally-cleavable moiety;

Y is independently absent, a bond or a continuation of a bond, or aspacer system which is comprised of 1 or more spacers;

M is independently: R or (RR′)-L′-(V′—(Y′—(Z)_(z′))_(q′))_(r′), wherein

R is a reactive moiety of Formula II or Formula Ib;

(RR′) is an addition product between Rand a complementary reactivemoiety R′;

L′ is independently absent, a bond or a continuation of a bond, or acarbon comprising framework of 1 to 200 atoms substituted at one or moreatoms, optionally, wherein the carbon comprising framework comprises alinear framework of 3 to 30 carbon atoms optionally substituted at oneor more atoms, optionally wherein the carbon comprising framework is alinear hydrocarbon, a symmetrically or asymmetrically branchedhydrocarbon, monosaccharide, disaccharide, linear or branchedoligosaccharide (asymmetrically branched or symmetrically branched),other natural linear or branched oligomers (asymmetrically branched orsymmetrically branched), or a dimer, trimer, or higher oligomer (linear,asymmetrically branched or symmetrically branched) resulting from anychain-growth or step-growth polymerization process;

V′ is independently absent, a bond or a continuation of a bond, anon-cleavable moiety or a conditionally-cleavable moiety;

Y′ is independently absent, a bond or a continuation of a bond, or aspacer system which is comprised of 1 or more spacers;

Z is independently a reactive group, a moiety that improves thepharmacokinetic properties, a therapeutic or diagnostic moiety, and eachZ is directly coupled to either Y or V when Y is absent, or L when bothY and V are absent; and

z′, q′ and r′ are each independently an integer selected from among 1,2, 3 or 4.

In one embodiment, the invention provides a method for conjugating amoiety of interest (Z) to an antibody, comprising the steps of:

a) providing an antibody having at least one acceptor glutamine residue;

b) reacting said antibody with a linking reagent of Formula Ib, in thepresence of a TGase, under conditions sufficient to obtain an antibodycomprising an acceptor glutamine linked (covalently) to a reactive group(R) via a lysine-based linker; and

(c) optionally, reacting (i) the antibody obtained in step b) with (ii)a compound comprising a moiety of interest (Z) and a reactive group (R′)capable of reacting with reactive group R, under conditions sufficientto obtain an antibody comprising an acceptor glutamine linked to amoiety of interest (Z) via a lysine-based linker is obtained.

In one embodiment, the invention provides a method for evaluating anantibody conjugate, the method comprising the steps of:

a) providing a first antibody composition of Formulae I-IV comprising afirst X, L, V, Y, L′, V′, Y′, (RR′) and/or Z moiety, wherein at least70%, 80% or 90% of the antibodies in said first antibody compositionhave (m) functionalized acceptor glutamine residues (Q) per antibody;

b) providing a second antibody composition of Formulae I-IV comprising asecond X, L, V, Y, L′, V′, Y′, (RR′) and/or Z moiety, wherein saidsecond antibody comprises at least one X, L, V, Y, L′, V′, Y′, (RR′)and/or Z moiety that differs from a respective X, L, V, Y, L′, V′, Y′,(RR′) and/or Z moiety of said first antibody, wherein at least 70%, 80%or 90% of the antibodies in said second antibody composition have (n)functionalized acceptor glutamine residues (Q) per antibody, andwherein; and

c) evaluating the first and second antibody compositions. In oneembodiment, n and m are equal. In one embodiment, m and n are 2. In oneembodiment, m and n are 4.

In one embodiment, the invention provides a kit comprising at least twoantibody compositions of Formula II or IVb, wherein the kit comprises afirst antibody composition and a second antibody composition in separatecontainers, wherein the second antibody composition comprises antibodieshaving at least one X, L, V, Y, L′, V′, Y′, (RR′) and/or Z moiety thatdiffers from a respective X, L, V, Y, L′, V′, Y′, (RR′) and/or Z moietyof said first antibody. In one embodiment, the antibodies of the firstand second antibody compositions share heavy and/or light chain aminoacid sequences.

In one embodiment of any of Formula I-IV, the antibody is a full lengthantibody. In one embodiment of any of Formula I-IV herein, the antibodyis an antibody fragment.

In one embodiment, the invention provides a pharmaceutical compositioncomprising an antibody or composition of any of Formula I-IV, and apharmaceutically acceptable carrier. In one embodiment, the inventionprovides a method of treating a disease comprising administering to amammal a composition of the invention.

In one embodiment of any of methods, linking reagents, compounds orantibodies herein, Z is a hydrophobic compound. In one embodiment Z isan organic compound comprising a molecular weight of at least 500 g/mol.In one embodiment Z is an organic compound comprising a molecular weightof at least 700 g/mol. In one embodiment Z is an organic compoundcomprising one or more cyclic groups, optionally a macrocycle,polycyclic or tricyclic group. In one embodiment Z is a negativelycharged compound. In one embodiment Z is a cytotoxic anti-cancer agent.In one embodiment Z is selected from the group consisting of taxanes,anthracyclines, camptothecins, epothilones, mytomycins, combretastatins,vinca alkaloids, nitrogen mustards, maytansinoids, calicheamycins,duocarmycins, tubulysins, dolastatins and auristatins, enediynes,pyrrolobenzodiazepines, and ethylenimines

In one embodiment, the invention provides method for preparing anantibody or antibody fragment comprising a moiety of interest (Z) boundthereto, comprising the steps of:

(a) immobilizing an antibody or antibody fragment comprising afunctionalized acceptor glutamine comprising a reactive moiety R ofFormula II or IVb on a solid support to provide an immobilized antibody,optionally comprising a step of applying an antibody-containing sampleto a solid support;

(b) reacting the immobilized antibody or antibody fragment of step (a)with a compound comprising a moiety-of-interest Z and a reaction partnerR′ and, optionally comprising a step of applying a compound comprising amoiety Z and a reactive group R′ to a solid support, to generate anantibody-moiety-of-interest conjugate. In one embodiment, the methodfurther comprises a washing step to remove any unreacted materials. Inone embodiment, the method further comprises a step of recoveringunreacted compound comprising a moiety Z and a reactive group R′ andre-applying said compound to the solid support to provide for highercompletion of the reaction between antibody comprising reactive group(R) and compound comprising reactive group (R′). In one embodiment, themethod further comprises a step of eluting immobilized antibodyconjugates from the solid support to provide antibody conjugatecompositions. In one embodiment, the compound comprising a moiety Z anda reactive group R′ is a compound of Formula III. In one embodiment, theantibody-moiety-of-interest conjugate obtained by the method is anantibody or antibody fragment of Formula II.

Reference to “Formulas I”, “Formula II”, “Formula III” or “Formula IV”,unless the context clearly indicates otherwise, designates all compoundsderived from such Formulas I to IV, including e.g., Formula I includesreference to Ia, Ib and/or Ic, Formula IV includes IVa and IVb.

Any of the methods of the invention can further be characterized ascomprising any step described in the application, including notably inthe “Detailed Description of the Invention”). The invention furtherrelates to an antibody obtainable by any of present methods. Theinvention further relates to pharmaceutical or diagnostic formulationsof the antibodies of the present invention. The invention furtherrelates to methods of using an antibody of Formula IV in a method oftreatment or diagnosis.

These and additional advantageous aspects and features of the inventionmay be further described elsewhere herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows reaction schemes for thio-maleimide additions, Staudingerligations, and Diels-Alder cycloadditions, where reactive groups oflinking reagents having a single reactive functionality combine withcomplementary reactive group attached to a therapeutic or diagnosticmoiety.

FIG. 2 shows reaction schemes for Diels-Alder cycloadditions and clickreactions where the reactive groups of linking reagents combine withcomplementary reactive group attached to an agent including atherapeutic, diagnostic, or other moiety.

FIG. 3 shows the preparation of an exemplary linking reagent, accordingto an embodiment of the invention, and its conjugation with a protein,where: V and Y are absent, R is a thiol (sulfhydryl) reactive group thatis ultimately generated from the S-acetyl protected thiol, SC(O)CH₃, ris 0; q is 0; z is 1; L is the two carbon comprising framework C(O)CH₂;X is NH; (C)_(n) is (CH₂)₅; and G is transformed from the (H₃C)₃COC(O)protecting group to H and ultimately to the amide upon conjugation of aglutamine residue of a protein.

FIG. 4 illustrates the preparation of various exemplary linkingreagents, according to various embodiments of the invention, with asingle S-acetyl protected thiol reactive group that can be prepared froman N-succinimidyl-5-acetylthioester reagent.

FIG. 5 illustrates the preparation of an exemplary linking reagent,according to an embodiment of the invention, and its conjugation with aprotein, where: V and Y are absent, R is an azide reactive group, r is0; q is 0; z is 1; L is the two carbon comprising framework C(O)CH₂; Xis NH; (C)_(n) is (CH₂)₅; and G is transformed from the (H₃C)₃COC(O)protecting group to H and ultimately to the amide upon conjugation of aglutamine residue of a protein.

FIG. 6 illustrates the preparation of various exemplary linkingreagents, according to embodiments of the invention, with a single azidereactive group that can be prepared from an N-succinimidyl-azidereagent.

FIG. 7 depicts the preparation of an exemplary linking reagent,according to an embodiment of the invention, and its conjugation with aprotein, where: V and Y are absent, R is an alkyne reactive group, r is0; q is 0; z is 1; L is a one carbon comprising framework CH₂; X is NH;(C)_(n) is (CH₂)₄—CH(CO₂H); and G is transformed from the (H₃C)₃COC(O)protecting group to H and ultimately to the amide upon conjugation of aglutamine residue of a protein.

FIG. 8 shows the preparation of an exemplary linking reagent, accordingto an embodiment of the invention, and its conjugation with a protein,where: R is a norbornene reactive group, r is 0; q is 0; z is 1; L isthe one carbon comprising framework C(O); X is NH; (C)_(n) is(CH₂)₄—CH(CO₂H); and G is transformed from the (H₃C)₃COC(O) protectinggroup to H and ultimately to the amide upon conjugation of a glutamineresidue of a protein.

FIG. 9 shows various examples of linking reagents according to theinvention.

FIGS. 10A and 10B show a general scheme for preparing conjugatedantibodies.

FIG. 11 shows a scheme for preparing an antibody conjugate from aS-acetyl-cadaverin linker of FIG. 3, where “R” in the figure is amoiety-of-interest Z.

FIG. 12 shows a scheme for preparing an antibody conjugate from anazide-cadaverin linker of FIG. 5, where “R” in the figure is amoiety-of-interest Z.

FIG. 13 shows a scheme for preparing an antibody conjugate from anorbornyl-cadaverin linker of FIG. 8, where “R” in the figure is amoiety-of-interest Z.

FIG. 14 shows a scheme for preparing an antibody conjugate fromalkyne-lysine linker of FIG. 7, where “R” in the figure is amoiety-of-interest Z.

FIG. 15 shows a scheme for preparing S-acetyl-protected cadaverinlinkers of different lengths (either n=1 or 5 carbons) as well as ashort thiol linker coupled to maleimide-DOTA.

FIGS. 16A, 16B and 16C show schemes for preparing linkers of theinvention.

FIGS. 17A and 17B show the deconvoluted mass spectra of chADC1 heavychain coupled to DOTA thiol linker 5 using either 1 U/mL (left) or 6U/mL BTG.

FIGS. 18A-18G shows optimized conditions for BTG coupling, including BTGconcentrations (18A), pH (18B and 18C), temperature (18D and 18E), andsubstrate stoichiometry (18F and 18G).

FIG. 19A shows improved enzymatic modification of deglycosylatedchimeric antibody heavy chain C6-DOTA linker by BTG, compared to C2-DOTAlinker.

FIG. 19B shows an ion chromatogram extract showing, after trypticdigest, a deamidated peptide including the biotin-modified glutamine atposition 295.

FIG. 19C shows the MS/MS spectrum and sequence confirmation of thespecific N297-deamidated tryptic peptide including the biotin-modifiedglutamine at position 295.

FIG. 20A shows the MS spectrum of chADCldgl coupled toC6-Maleimide-vc-PAB-MMAF. FIG. 20B shows the MS spectrum of chADC1N297Scoupled to C6-Maleimide-vc-PAB-MMAF.

FIGS. 21A and 21B show improved enzymatic modification of N297S chimericantibody heavy chain with C6-DOTA linker by BTG, compared to C6-DOTAlinker on PNGaseF-deglycosylated antibody.

FIGS. 22A and 22B show the deconvoluted mass spectra of chADC1 heavychain coupled to the short (left) and long (right) thiol linker,compounds 4a and 4b. The FIG. 22A spectrum shows the protected shortlinker compound 4a and the FIG. 22B spectrum shows deprotected longlinker 4b.

FIG. 23A shows LC-MS analysis of untagged nanobody incubated with BTG(top) or BTG and biotin-cadaverin (bottom).FIG. 23B shows LC-MS analysisof myc-tagged nanobody incubated with BTG (top) or BTG andbiotin-cadaverin (bottom). FIG. 23C shows LC-MS analysis of myc-taggeddimeric affibody incubated with BTG only (top) or BTG andbiotin-cadaverin (middle) or BTG and dansyl-cadaverin (bottom).

DETAILED DESCRIPTION OF THE INVENTION Introduction

According to the invention, the functionalization of antibodies issite-specific and occurs via, respectively between a primary amine (e.g.of a lysine or lysine-like moiety) and an acceptor glutamine residue ofan antibody by transglutaminase.

The inventors now present a convenient method for the site-specificfunctionalization by large chemical molecules (e.g., cytotoxic drugssuch as duocarmycins, auristatins, calcheamycins that are naturalproduct derivatives or polymers) of immunoglobulins under nearphysiological conditions. The enzymatic activity of the transglutaminasefamily catalyzes an acyl transfer reaction between the γ-carboxamidegroups of peptide-bound glutamine residues and various primary amines orE-amino groups of lysine residues, thus forming isopeptidic bonds whichare stable and resistant to chemical, enzymatic, and physicaldegradation. The function of TGases can be described as incorporation ofalkylamine derivatives into specific glutamine residues or vice versa.This specificity has been recognized before and has already been appliedsuccessfully for different purposes.

DEFINITIONS

As used in the specification, “a” or “an” may mean one or more. As usedin the claim(s), when used in conjunction with the word “comprising”,the words “a” or “an” may mean one or more than one. As used herein“another” may mean at least a second or more.

Where “comprising” is used, this can be replaced by “consistingessentially of”, or by “consisting of”.

The term “transglutaminase”, used interchangeably with “TGase” or “TG”,refers to an enzyme capable of cross-linking proteins through anacyl-transfer reaction between the γ-carboxamide group of peptide-boundglutamine and the E-amino group of a lysine or a structurally relatedprimary amine such as amino pentyl group, e.g. a peptide-bound lysine,resulting in a ε-(γ-glutamyl)lysine isopeptide bond. TGases include,inter alia, bacterial transglutaminase (BTG) such as the enzyme havingEC reference EC 2.3.2.13 (protein-glutamine-γ-glutamyltransferase).

The term “acceptor glutamine residue”, when referring to a glutamineresidue of an antibody, means a glutamine residue that is recognized bya TGase and can be cross-linked by a TGase through a reaction betweenthe glutamine and a lysine or a structurally related primary amine suchas amino pentyl group. Preferably the acceptor glutamine residue is asurface-exposed glutamine residue.

The term “antibody” herein is used in the broadest sense andspecifically includes full-length monoclonal antibodies, polyclonalantibodies, multispecific antibodies (e.g., bispecific antibodies), andantibody fragments, so long as they exhibit the desired biologicalactivity. Various techniques relevant to the production of antibodiesare provided in, e.g., Harlow, et al., ANTIBODIES: A LABORATORY MANUAL,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1988).

An “antibody fragment” comprises a portion of a full-length antibody,preferably antigen-binding or variable regions thereof. Examples ofantibody fragments include Fab, Fab′, F(ab)₂, F(ab′)₂, F(ab)₃, Fv(typically the VL and VH domains of a single arm of an antibody),single-chain Fv (scFv), dsFv, Fd fragments (typically the VH and CH1domain), and dAb (typically a VH domain) fragments; VH, VL, VhH, andV-NAR domains; minibodies, diabodies, triabodies, tetrabodies, and kappabodies (see, e.g., Ill et al., Protein Eng 1997; 10: 949-57); camel IgG;IgNAR; and multispecific antibody fragments formed from antibodyfragments, and one or more isolated CDRs or a functional paratope, whereisolated CDRs or antigen-binding residues or polypeptides can beassociated or linked together so as to form a functional antibodyfragment. Various types of antibody fragments have been described orreviewed in, e.g., Holliger and Hudson, Nat Biotechnol 2005; 23,1126-1136; WO2005040219, and published U.S. Patent Applications20050238646 and 20020161201.

By “variable region” as used herein is meant the region of an antibodythat comprises one or more Ig domains substantially encoded by any ofthe VL (including Vkappa and Vlambda) and/or VH genes that make up thelight chain (including kappa and lambda) and heavy chain immunoglobulingenetic loci respectively. A light or heavy chain variable region (VLand VH) consists of a “framework” or “FR” region interrupted by threehypervariable regions referred to as “complementarity determiningregions” or “CDRs”. The extent of the framework region and CDRs havebeen precisely defined, for example as in Kabat (see “Sequences ofProteins of Immunological Interest,” E. Kabat et al., U.S. Department ofHealth and Human Services, (1983)), and as in Chothia. The frameworkregions of an antibody, that is the combined framework regions of theconstituent light and heavy chains, serves to position and align theCDRs, which are primarily responsible for binding to an antigen.

By “constant region” of an antibody as defined herein is meant theregion of the antibody that is encoded by one of the light or heavychain immunoglobulin constant region genes. By “constant light chain” or“light chain constant region” as used herein is meant the region of anantibody encoded by the kappa (Ckappa) or lambda (Clambda) light chains.The constant light chain typically comprises a single domain, and asdefined herein refers to positions 108-214 of Ckappa, or Clambda,wherein numbering is according to the EU index of Kabat (Kabat et al.,1991, Sequences of Proteins of Immunological Interest, 5th Ed., UnitedStates Public Health Service, National Institutes of Health, Bethesda)and/or Edelman, G. M. et al., Proc. Natl. Acad. USA, 63, 78-85 (1969).By “constant heavy chain” or “heavy chain constant region” as usedherein is meant the region of an antibody encoded by the mu, delta,gamma, alpha, or epsilon genes to define the antibody's isotype as IgM,IgD, IgG, IgA, or IgE, respectively. For full length IgG antibodies, theconstant heavy chain, as defined herein, refers to the N-terminus of theCH1 domain to the C-terminus of the CH3 domain, thus comprisingpositions 118-447. Unless indicated otherwise, numbering within theconstant region is according to the EU index of Kabat (1991) and/orEdelman, G. M. et al., Proc. Natl. Acad. USA, 63, 78-85 (1969).

By “Fab” or “Fab region” as used herein is meant the polypeptide thatcomprises the VH, CH₁, VL, and CL immunoglobulin domains. Fab may referto this region in isolation, or this region in the context of a fulllength antibody, antibody fragment or Fab fusion protein, or any otherantibody embodiments as outlined herein.

By “Fv” or “Fv fragment” or “Fv region” as used herein is meant apolypeptide that comprises the VL and VH domains of a single antibody.

By “Fc”, “Fc domain” or “Fc region”, as used herein is meant thepolypeptide comprising the constant region of an antibody excluding thefirst constant region immunoglobulin domain. Thus Fc refers to the lasttwo constant region immunoglobulin domains of IgA, IgD, and IgG, and thelast three constant region immunoglobulin domains of IgE and IgM, andthe flexible hinge N-terminal to these domains For IgA and IgM, Fc mayinclude the J chain. For IgG, as illustrated in FIG. 1, Fc comprisesimmunoglobulin domains Cγ2 and Cγ3 and the hinge between Cγ1 and Cγ2.Although the boundaries of the Fc region may vary, the human IgG heavychain Fc region is usually defined to comprise residues C226 or P230 toits carboxyl-terminus, wherein the numbering is according to the EUindex as in Kabat. Fc may refer to this region in isolation, or thisregion in the context of an Fc polypeptide, as described below.

By “full length antibody” as used herein is meant the structure thatconstitutes the natural biological form of an antibody, includingvariable and constant regions. For example, in most mammals, includinghumans and mice, the full length antibody of the IgG isotype is atetramer and consists of two identical pairs of two immunoglobulinchains, each pair having one light and one heavy chain, each light chaincomprising immunoglobulin domains VL and CL, and each heavy chaincomprising immunoglobulin domains VH, Cγ1, Cγ2, and Cγ3. In somemammals, for example in camels and llamas, IgG antibodies may consist ofonly two heavy chains, each heavy chain comprising a variable domainattached to the Fc region.

By “amino acid modification” herein is meant an amino acid substitution,insertion, and/or deletion in a polypeptide sequence. The preferredamino acid modification herein is a substitution. By “amino acidmodification” herein is meant an amino acid substitution, insertion,and/or deletion in a polypeptide sequence. By “amino acid substitution”or “substitution” herein is meant the replacement of an amino acid at agiven position in a protein sequence with another amino acid. Forexample, the substitution Y50W refers to a variant of a parentpolypeptide, in which the tyrosine at position 50 is replaced withtryptophan. A “variant” of a polypeptide refers to a polypeptide havingan amino acid sequence that is substantially identical to a referencepolypeptide, typically a native or “parent” polypeptide. The polypeptidevariant may possess one or more amino acid substitutions, deletions,and/or insertions at certain positions within the native amino acidsequence.

“Conservative” amino acid substitutions are those in which an amino acidresidue is replaced with an amino acid residue having a side chain withsimilar physicochemical properties. Families of amino acid residueshaving similar side chains are known in the art, and include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine), beta-branchedside chains (e.g., threonine, valine, isoleucine) and aromatic sidechains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

An “isolated” molecule is a molecule that is the predominant species inthe composition wherein it is found with respect to the class ofmolecules to which it belongs (i.e., it makes up at least about 50% ofthe type of molecule in the composition and typically will make up atleast about 70%, at least about 80%, at least about 85%, at least about90%, at least about 95%, or more of the species of molecule, e.g.,peptide, in the composition). Commonly, a composition of an antibodymolecule will exhibit 98%, 98%, or 99% homogeneity for antibodymolecules in the context of all present peptide species in thecomposition or at least with respect to substantially active peptidespecies in the context of proposed use.

In the context of the present invention, “treatment” or “treating”refers to preventing, alleviating, managing, curing or reducing one ormore symptoms or clinically relevant manifestations of a disease ordisorder, unless contradicted by context. For example, “treatment” of apatient in whom no symptoms or clinically relevant manifestations of adisease or disorder have been identified is preventive or prophylactictherapy, whereas “treatment” of a patient in whom symptoms or clinicallyrelevant manifestations of a disease or disorder have been identifiedgenerally does not constitute preventive or prophylactic therapy.

The term “reactive moiety” herein refers to a moiety that can be coupledwith another moiety without prior activation or transformation.

The term “protecting group” refers to a group that temporarily protectsor blocks, i.e., intended to prevent from reacting, a functional group,e.g., an amino group, a hydroxyl group, or a carboxyl group, during thetransformation of a first molecule to a second molecule.

The phrase “moiety that improves the pharmacokinetic properties”, whenreferring to a compound (e.g. an antibody) refers to a moiety thatchanges the pharmacokinetic properties of the one or more moieties Z insuch a way that a better therapeutic or diagnostic effect can beobtained. The moiety can for example increase the water solubility,increase the circulation time, or reduce immunogenicity.

The phrase “linking group” refers to a structural element of a compoundthat links one structural element of said compound to one or more otherstructural elements of said same compound.

The phrase “a number representing degree of branching” is used to denotethat the subscript number next to a closing bracket represents how manyunits of the moiety within the brackets are attached to the moietydirectly to the left of the corresponding opening bracket For example,A-(B)_(b) with b being a number representing a degree of branching meansthat b units B are all directly attached to A This means that when b is2, the formula reduces to B-A-B.

The phrase “a number representing degree of polymerization” is used todenote that the subscript number next to a closing bracket representshow many units of the moiety within the brackets are connected to eachother. For example, A-(B)₁, with b being a number representing a degreeof polymerization means that when b is 2, the formula reduces to A-B—B.

The term “identity” or “identical”, when used in a relationship betweenthe sequences of two or more polypeptides, refers to the degree ofsequence relatedness between polypeptides, as determined by the numberof matches between strings of two or more amino acid residues.“Identity” measures the percent of identical matches between the smallerof two or more sequences with gap alignments (if any) addressed by aparticular mathematical model or computer program (i.e., “algorithms”).Identity of related polypeptides can be readily calculated by knownmethods. Such methods include, but are not limited to, those describedin Computational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York, 1988; Biocomputing: Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis ofSequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., HumanaPress, New Jersey, 1994; Sequence Analysis in Molecular Biology, vonHeinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M.and Devereux, J., eds., M. Stockton Press, New York, 1991; and Carilloet al., SIAM J. Applied Math. 48, 1073 (1988).

Preferred methods for determining identity are designed to give thelargest match between the sequences tested. Methods of determiningidentity are described in publicly available computer programs.Preferred computer program methods for determining identity between twosequences include the GCG program package, including GAP (Devereux etal., Nucl. Acid. Res. 12, 387 (1984); Genetics Computer Group,University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA(Altschul et al., J. Mol. Biol. 215, 403-410 (1990)). The BLASTX programis publicly available from the National Center for BiotechnologyInformation (NCBI) and other sources (BLAST Manual, Altschul et al.NCB/NLM/NIH Bethesda, Md. 20894; Altschul et al., supra). The well knownSmith Waterman algorithm may also be used to determine identity.

As used herein, “alkyl” refers to a straight or branched hydrocarbonchain that comprises a fully saturated (no double or triple bonds)hydrocarbon group. The alkyl group may have, for example, 1 to 20 carbonatoms (whenever it appears herein, a numerical range such as “1 to 20”refers to each integer in the given range; e.g., “1 to 20 carbon atoms”means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms,3 carbon atoms, etc., up to and including 20 carbon atoms, although thepresent definition also covers the occurrence of the term “alkyl” whereno numerical range is designated). The alkyl group of the compounds maybe designated as “C₁-C₄ alkyl” or similar designations. By way ofexample only, “C₁-C₄ alkyl” indicates that there are one to four carbonatoms in the alkyl chain, i.e., the alkyl chain is selected from methyl,ethyl, propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, and t-butyl.Typical alkyl groups include, but are in no way limited to, methyl,ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl andhexyl. The alkyl group may be substituted or unsubstituted.

As used herein, the term “heteroalkyl” refers to a straight or branchedalkyl group that contains one or more heteroatoms, that is, an elementother than carbon (including but not limited to oxygen, sulfur,nitrogen, phosphorus) in place of one or more carbon atoms.

Whenever a group is described as being “substituted” that groupsubstituted with one or more of the indicated substituents. If nosubstituents are indicated, it is meant that the indicated “substituted”group may be substituted with one or more group(s) individually andindependently selected from alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, cycloalkynyl, heteroalkyl, aryl, heteroaryl,heteroalicyclyl, aralkyl, heteroaralkyl, (heteroalicyclyl)alkyl,hydroxy, alkoxy, aryloxy, acyl, mercapto, alkylthio, arylthio, cyano,halogen, thiocarbonyl, carbamyl, thiocarbamyl, amido, sulfonamido,sulfonamido, carboxy, isocyanato, thiocyanato, isothiocyanato, nitro,silyl, sulfenyl, sulfinyl, sulfonyl, haloalkyl, haloalkoxy,trihalomethanesulfonyl, trihalomethanesulfonamido, an amino, amono-substituted amino group and a di-substituted amino group, andprotected derivatives thereof.

Where the number of substituents is not specified (e.g. haloalkyl),there may be one or more substituents present. For example “haloalkyl”may include one or more of the same or different halogens. As anotherexample, “C₁-C₃ alkoxyphenyl” may include one or more of the same ordifferent alkoxy groups containing one, two or three atoms.

The term “pharmaceutically acceptable salt” refers to a salt of acompound that does not cause significant irritation to an organism towhich it is administered and does not abrogate the biological activityand properties of the compound. In some embodiments, the salt is an acidaddition salt of the compound. Pharmaceutical salts can be obtained byreacting a compound with inorganic acids such as hydrohalic acid (e.g.,hydrochloric acid or hydrobromic acid), sulfuric acid, nitric acid andphosphoric acid. Pharmaceutical salts can also be obtained by reacting acompound with an organic acid such as aliphatic or aromatic carboxylicor sulfonic acids, for example formic, acetic, succinic, lactic, malic,tartaric, citric, ascorbic, nicotinic, methanesulfonic, ethanesulfonic,p-toluensulfonic, salicylic or naphthalenesulfonic acid. Pharmaceuticalsalts can also be obtained by reacting a compound with a base to form asalt such as an ammonium salt, an alkali metal salt, such as a sodium ora potassium salt, an alkaline earth metal salt, such as a calcium or amagnesium salt, a salt of organic bases such as dicyclohexylamine,N-methyl-D-glucamine, tris(hydroxymethyl)methylamine, C₁-C₇ alkylamine,cyclohexylamine, triethanolamine, ethylenediamine, and salts with aminoacids such as arginine and lysine.

Producing Antibodies

Antibodies may be produced by a variety of techniques known in the art.Typically, they are produced by immunization of a non-human animal,preferably a mouse, with an immunogen comprising a polypeptide, or afragment or derivative thereof, typically an immunogenic fragment, forwhich it is desired to obtain antibodies (e.g. a human polypeptide). Thestep of immunizing a non-human mammal with an antigen may be carried outin any manner well known in the art for stimulating the production ofantibodies in a mouse (see, for example, E. Harlow and D. Lane,Antibodies: A Laboratory Manual., Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. (1988), the entire disclosure of which isherein incorporated by reference). Other protocols may also be used aslong as they result in the production of B cells expressing an antibodydirected to the antigen used in immunization. Lymphocytes from anon-immunized non-human mammal may also be isolated, grown in vitro, andthen exposed to the immunogen in cell culture. The lymphocytes are thenharvested and the fusion step described below is carried out. Forpreferred monoclonal antibodies, the next step is the isolation ofsplenocytes from the immunized non-human mammal and the subsequentfusion of those splenocytes with an immortalized cell in order to forman antibody-producing hybridoma. The hybridoma colonies are then assayedfor the production of antibodies that specifically bind to thepolypeptide against which antibodies are desired. The assay is typicallya colorimetric ELISA-type assay, although any assay may be employed thatcan be adapted to the wells that the hybridomas are grown in. Otherassays include radioimmunoassays or fluorescence activated cell sorting.The wells positive for the desired antibody production are examined todetermine if one or more distinct colonies are present. If more than onecolony is present, the cells may be re-cloned and grown to ensure thatonly a single cell has given rise to the colony producing the desiredantibody. After sufficient growth to produce the desired monoclonalantibody, the growth media containing monoclonal antibody (or theascites fluid) is separated away from the cells and the monoclonalantibody present therein is purified. Purification is typically achievedby gel electrophoresis, dialysis, chromatography using protein A orprotein G-Sepharose, or an anti-mouse Ig linked to a solid support suchas agarose or Sepharose beads (all described, for example, in theAntibody Purification Handbook, Biosciences, publication No. 18-1037-46,Edition AC, the disclosure of which is hereby incorporated byreference).

Human antibodies may also be produced by using, for immunization,transgenic animals that have been engineered to express a human antibodyrepertoire (Jakobovitz et Nature 362 (1993) 255), or by selection ofantibody repertoires using phage display methods. For example, aXenoMouse (Abgenix, Fremont, Calif.) can be used for immunization. AXenoMouse is a murine host according to this invention that has had itsimmunoglobulin genes replaced by functional human immunoglobulin genes.Thus, antibodies produced by this mouse or in hybridomas made from the Bcells of this mouse, are already humanized. The XenoMouse is describedin U.S. Pat. No. 6,162,963, which is herein incorporated in its entiretyby reference.

Antibodies may also be produced by selection of combinatorial librariesof immunoglobulins, as disclosed for instance in (Ward et al. Nature,341 (1989) p. 544, the entire disclosure of which is herein incorporatedby reference). Phage display technology (McCafferty et al (1990) Nature348:552-553) can be used to produce antibodies from immunoglobulinvariable (V) domain gene repertoires from unimmunized donors. See, e.g.,Griffith et al (1993) EMBO J. 12:725-734; U.S. Pat. No. 5,565,332; U.S.Pat. No. 5,573,905; U.S. Pat. No. 5,567,610; U.S. Pat. No. 5,229,275).When combinatorial libraries comprise variable (V) domain generepertoires of human origin, selection from combinatorial libraries willyield human antibodies.

Additionally, a wide range of antibodies are available in the scientificand patent literature, including DNA and/or amino acid sequences, orfrom commercial suppliers. Examples of antibodies include antibodiesthat recognize an antigen expressed by a target cell that is to beeliminated, for example a proliferating cell or a cell contributing to apathology. Examples include antibodies that recognize tumor antigens,microbial (e.g. bacterial) antigens or viral antigens. Other examplesinclude antigens present on immune cells that are contributing toinflammatory or autoimmune disease, including rejection of transplantedtissue (e.g. antigens present on T cells (CD4 or CD8 T cells).

Antibodies will typically be directed to a pre-determined antigen. Asused herein, the term “bacterial antigen” includes, but is not limitedto, intact, attenuated or killed bacteria, any structural or functionalbacterial protein or carbohydrate, or any peptide portion of a bacterialprotein of sufficient length (typically about 8 amino acids or longer)to be antigenic. Examples include gram-positive bacterial antigens andgram-negative bacterial antigens. In preferred embodiments of thepresent invention the bacterial antigen is derived from a bacteriumselected from the group consisting of Helicobacter species, inparticular Helicobacter pyloris; Borelia species, in particular Boreliaburgdorferi; Legionella species, in particular Legionella pneumophilia;Mycobacteria s species, in particular M. tuberculosis, M. avium, M.intracellulare, M. kansasii, M. gordonae; Staphylococcus species, inparticular Staphylococcus aureus; Neisseria species, in particular N.gonorrhoeae, N. meningitidis; Listeria species, in particular Listeriamonocytogenes; Streptococcus species, in particular S. pyogenes, S.agalactiae; S. faecalis; S. bovis, S. pneumonias; anaerobicStreptococcus species; pathogenic Campylobacter species; Enterococcusspecies; Haemophilus species, in particular Haemophilus influenzue;Bacillus species, in particular Bacillus anthracis; Corynebacteriumspecies, in particular Corynebacterium diphtheriae; Erysipelothrixspecies, in particular Erysipelothrix rhusiopathiae; Clostridiumspecies, in particular C. perfringens, C. tetani; Enterobacter species,in particular Enterobacter aerogenes, Klebsiella species, in particularKlebsiella 1 S. pneumoniae, Pasturella species, in particular Pasturellamultocida, Bacteroides species; Fusobacterium species, in particularFusobacterium nucleatum; Streptobacillus species, in particularStreptobacillus moniliformis; Treponema species, in particular Treponemapertenue; Leptospira; pathogenic Escherichia species; and Actinomycesspecies, in particular Actinomyces israelli.

As used herein, the term “viral antigen” includes, but is not limitedto, intact, attenuated or killed whole virus, any structural orfunctional viral protein, or any peptide portion of a viral protein ofsufficient length (typically about 8 amino acids or longer) to beantigenic. Sources of a viral antigen include, but are not limited toviruses from the families. Retroviridae (e.g., human immunodeficiencyviruses, such as HIV-1 (also referred to as HTLV-III, LAV orHTLV-III/LAV, or HIV-III; and other isolates, such as HIV-LP;Picornaviridae (e.g., polio viruses, hepatitis A virus; enteroviruses,human Coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g.,strains that cause gastroenteritis); Togaviridae (e.g., equineencephalitis viruses, rubella viruses); Flaviviridae (e.g., dengueviruses, encephalitis viruses, yellow fever viruses); Coronaviridae(e.g., coronaviruses); Rhabdoviridae (e.g., vesicular stomatitisviruses, rabies viruses); Filoviridae (e.g., ebola viruses);Paramyxoviridae (e.g., parainfluenza viruses, mumps virus, measlesvirus, respiratory syncytial virus); Orthomyxoviridae (e.g., influenzaviruses); Bunyaviridae (e.g., Hantaan viruses, bunya viruses,phleboviruses and Nairo viruses); Arenaviridae (hemorrhagic feverviruses); Reoviridae (e.g., reoviruses, orbiviruses and rotaviruses);Bornaviridae; Hepadnaviridae (Hepatitis B virus); Parvoviridae(parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses);Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus(HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpesvirus; Poxyiridae (variola viruses, vaccinia viruses, pox viruses); andIridoviridae (e.g., African swine fever virus); and unclassified viruses(e.g., the agent of delta hepatitis (thought to be a defective satelliteof hepatitis B virus), Hepatitis C; Norwalk and related viruses, andastroviruses). Alternatively, a viral antigen may be producedrecombinantly.

As used herein, the terms “cancer antigen” and “tumor antigen” are usedinterchangeably and refer to antigens (e.g., carbohydrates,polypeptides, or any peptide of sufficient length (typically about 8amino acids or longer) to be antigenic) that are differentiallyexpressed by cancer cells and can thereby be exploited in order totarget cancer cells. Cancer antigens are antigens which can potentiallystimulate apparently tumor-specific immune responses. Some of theseantigens are encoded, although not necessarily expressed, by normalcells. These antigens can be characterized as those which are normallysilent (i.e., not expressed) in normal cells, those that are expressedonly at certain stages of differentiation and those that are temporallyexpressed such as embryonic and fetal antigens. Other cancer antigensare encoded by mutant cellular genes, such as oncogenes (e.g., activatedras oncogene), suppressor genes (e.g., mutant p53), fusion proteinsresulting from internal deletions or chromosomal translocations. Stillother cancer antigens can be encoded by viral genes such as thosecarried on RNA and DNA tumor viruses.

The cancer antigens are usually normal cell surface antigens which areeither over-expressed or expressed at abnormal times. Ideally the targetantigen is expressed only on proliferative cells (preferably tumourcells), however this is rarely observed in practice. As a result, targetantigens are usually selected on the basis of differential expressionbetween proliferative and healthy tissue. Antibodies have been raised totarget specific tumour related antigens including: Cripto, CD4, CD20,CD30, CD19, CD33, Glycoprotein NMB, CanAg, Her2 (ErbB2/Neu), CD56(NCAM), CD22 (Siglec2), CD33 (Siglec3), CD79, CD138, CD171, PSCA, PSMA(prostate specific membrane antigen), BCMA, CD52, CD56, CD80, CD70,E-selectin, EphB2, Melanotransferin, Mud 6 and TMEFF2. Examples ofcancer antigens also include B7-H3, B7-H4, B7-H6, PD-L1, MAGE,MART-1/Melan-A, gp100, adenosine deaminase-binding protein (ADAbp),cyclophilin b, colorectal associated antigen (CRC)-0017-1A/GA733,carcinoembryonic antigen (CEA) and its immunogenic epitopes CAP-1 andCAP-2, etv6, aml1, prostate specific antigen (PSA), T-cellreceptor/CD3-zeta chain, MAGE-family of tumor antigens, GAGE-family oftumor antigens, BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, MUC family,VEGF, VEGF receptors, PDGF, TGF-alpha, EGF, EGF receptor, a member ofthe human EGF-like receptor family such as HER-2/neu, HER-3, HER-4 or aheterodimeric receptor comprised of at least one HER subunit, gastrinreleasing peptide receptor antigen, Muc-1, CAl25, αvβ3 integrins, α5β1integrins, αIIbβ3-integrins, PDGF beta receptor, SVE-cadherin, IL-8,hCG, IL-6, IL-6 receptor, IL-15, α-fetoprotein, E-cadherin, α-catenin,β-catenin and γ-catenin, p120ctn, PRAME, NY-ESO-1, cdc27, adenomatouspolyposis coli protein (APC), fodrin, Connexin 37, Ig-idiotype, p15,gp75, GM2 and GD2 gangliosides, viral products such as humanpapillomavirus proteins, imp-1, HA, EBV-encoded nuclear antigen(EBNA)-1, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40),SSX-1, SSX-4, SSX-5, SCP-1 and CT-7, and c-erbB-2, although this is notintended to be exhaustive.

DNA encoding an antibody of interest can be placed in an appropriateexpression vector for transfection into an appropriate host. The host isthen used for the recombinant production of the antibody, or variantsthereof, such as a humanized version of that monoclonal antibody, activefragments of the antibody, chimeric antibodies comprising the antigenrecognition portion of the antibody, or versions comprising a detectablemoiety.

In certain embodiments, the DNA of a hybridoma or other cell producingan antibody can be modified prior to insertion into an expressionvector, for example, by substituting the coding sequence for humanheavy- and light-chain constant domains in place of the homologousnon-human sequences (e.g., Morrison et al., PNAS pp. 6851 (1984)), or bycovalently joining to the immunoglobulin coding sequence all or part ofthe coding sequence for a non-immunoglobulin polypeptide. In thatmanner, “chimeric” or “hybrid” antibodies are prepared that have thebinding specificity of the original antibody. Typically, suchnon-immunoglobulin polypeptides are substituted for the constant domainsof an antibody of the invention.

Humanized antibodies can also be prepared. Humanized antibodies aretypically specific chimeric immunoglobulins, immunoglobulin chains orfragments thereof (such as Fv, Fab, Fab′, F (ab′)2, “dab”, or otherantigen-binding subsequences of antibodies) which contain minimalsequence derived from the murine immunoglobulin. For the most part,humanized antibodies are human immunoglobulins (recipient antibody) inwhich residues from a complementary-determining region (CDR) of therecipient are replaced by residues from a CDR of the original antibody(the parent or donor antibody) while maintaining the desiredspecificity, affinity, and capacity of the original antibody. The CDRsof the parent antibody, some or all of which are encoded by nucleicacids originating in a non-human organism, are grafted in whole or inpart into the beta-sheet framework of a human antibody variable regionto create an antibody, the specificity of which is determined by theengrafted CDRs. The creation of such antibodies is described in, e.g.,WO 92/11018, Jones, 1986, Nature 321:522-525, Verhoeyen et al., 1988,Science 239:1534-1536.

The antibody may or may not further comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details see Jones et al., Nature, 321, pp.522 (1986); Reichmann et al, Nature, 332, pp. 323 (1988); Presta, Curr.Op. Struct. Biol., 2, pp. 593 (1992); Verhoeyen et Science, 239, pp.1534; and U.S. Pat. No. 4,816,567, the entire disclosures of which areherein incorporated by reference.

Wild-type full-length IgG antibodies of human isotype will possess aconserved acceptor glutamine at residue 295 of the heavy chain whichwhen in non-glycosylated form will be accessible to a TGase andtherefore reactive with a compound of Formula I in the presence of aTGase, under suitable conditions, to form a conjugate from the antibodyand the compound of Formula II. The antibody will lack glycosylation atthe asparagine at residue 297 of the heavy chain

Additional or alternative sites reactive with a compound of Formula I inthe presence of a TGase can be created by engineering the antibodies.The compounds of the invention include glutamine engineered antibodieswhere one or more amino acids of a wild-type or parent antibody arereplaced with (substituted by) a glutamine amino acid, or where aglutamine residue, optionally together with other amino acid residues,is introduced or added to a wild-type or parent antibody (e.g. whereinthe glutamine residue is added to an antibody fragment).

It should be noted that a single site mutation that provides a glutaminethat is accessible to a TGase may yield more than one engineeredglutamine residue that can be conjugated if the antibody comprises morethan one engineered chain. For example, a single site mutation willyield two engineered glutamine residues in a tetrameric IgG due to thedimeric nature of the IgG antibody. The engineered glutamine residueswill be in addition to any acceptor glutamine already present in anantibody, if any. The glutamine amino acid residues that are reactive,in the presence of a TGase under suitable conditions, with a compound ofFormula I may be located in the heavy chain, typically in the constantdomain.

In one embodiment, an asparagine at amino acid position 297 (EU Index)is substituted with a glutamine residue. The antibody will have aconstant region with a N297Q substitution (a N297Q variant antibody). Anantibody having a N297Q substitution and a glutamine at residue 295 (EUIndex) will therefore have two acceptor glutamines and thus twoconjugation sites per heavy chain. In tetravalent form will thereforehave four conjugates per antibody. Such an antibody will be particularlywell adapted for use in conjunction with the multi-step method of theinvention; the antibody can be reacted with a compound of Formula Ib orIc to form an antibody of Formula II.

In one embodiment, an asparagine at amino acid position 297 issubstituted with a non-glutamine residue. The antibody will have aconstant region with a or Q295X (e.g., Q295X/N297Q), N297X, S298X and/orT299X substitution (a Q295X, N297X, S298X and/or T299X variantantibody), wherein X is any amino acid (other than a glutamine or theresidue Q, N, S or T naturally present at the respective 297, 298 or 299residue), optionally wherein the substitution is a conservativesubstitution. An antibody having a Q295X will be understood to have anintroduced glutamine at a different position, e.g., the antibody willalso have a N297Q substitution. Such an antibody, when comprising aglutamine at position 295 (a glutamine is naturally present in humanconstant regions at position 295) but no other acceptor glutamineresidues, will have two conjugates per antibody when the antibodycomprises two heavy chains. Such an antibody will additionally have theadvantage of being devoid of closely spaced acceptor glutamine residuessuch as could be present in an antibody having an acceptor glutamine atpositions 295 and 297, where the closely spaced acceptor residues whenfunctionalized with a linker comprising a reactive moiety (R) could leadto unwanted reactions between unprotected reactive group R. Suchunwanted reactions between (R) groups would make them unavailable forreactions with a compound of Formula III and a resulting composition ofantibodies of Formula IVb would have increased heterogeneity.

As shown herein, TGase provides limited ability to directly (in a singlecoupling reaction) couple linkers comprising large and/or hydrophobicmoieties (e.g., V, Y or Z) to PNGaseF-deglycosylated antibodies. PNGaseFtreatment of N297-glycosylated antibodies leads to the deamidation ofthe asparagine such that an aspartic acid residue is formed at position297 (EU numbering) following removal of the N-linked glycan (residue 297is at the +2 position relative to the glutamine at position 295 of theheavy chain in human IgG antibodies). This negatively charged asparticacid residue is believed to affect the ability of TGase to couplelinkers comprising large and/or hydrophobic moieties, and resultingantibody-linker conjugate compositions are heterogeneous, characterizedby antibodies having non-functionalized acceptor glutamines. However, bymodifying the antibody such that the residue at the +2 position(C-terminal to the acceptor glutamine), TGase becomes able tofunctionalize all acceptor glutamines on substantially all antibodies ina composition with linkers comprising large and/or hydrophobic moieties.Consequently, antibodies comprising a functionalized acceptor glutamineresidue flanked at position +2 by a non-aspartic acid residue can beused as an advantageous substrate for TGase-mediated conjugation oflinkers comprising large and/or hydrophobic moieties, particularly whena one-step conjugation reaction scheme is used.

An advantageous approach for preparing conjugated antibodies will thusinvolve providing as starting materials antibodies lacking N297-linkedglycosylation (such N-linked glycosylation interferes with TGasecoupling onto residue 295), wherein the +2 position relative to anacceptor glutamine is a non-aspartic acid residue. The residue at the +2position can be any suitable amino acid that permits efficientTGase-mediated conjugation. Optionally, the residue at the +2 positionis a non-negatively charged amino acid, e.g. any electrically neutralamino acid, a serine, etc. Optionally, the residue at the +2 position isselected from the group consisting of: amino acids with positivelycharged side chains, amino acids with polar uncharged side chains, andamino acids with hydrophobic side chains

One approach for preparing antibodies comprising a functionalizedacceptor glutamine residue flanked at position +2 by a non-aspartic acidresidue is to prepare antibodies having an asparagine at position 297but lacking N-linked glycosylation by a suitable method that does nottransform the asparagine at residue 297 to an aspartic acid. Forexample, antibodies can be produced in a host cell (e.g. a prokaryoticcell, E. coli) that does not yield N-glycosylated antibodies. Suchantibodies will typically have a glutamine in their heavy chain atposition 295 and a non-glycosylated asparagine at position 297, i.e. theresidue at the +2 position relative to an acceptor glutamine is anasparagine.

Preparing antibodies comprising a functionalized acceptor glutamineresidue flanked at the +2 position by a non-aspartic acid residue canalso be achieved by protein engineering. For example, an antibody havinga glutamine naturally present at heavy chain residue 295 (EU numbering)can comprise a modification at residue 297 such that the asparagine isdeleted or replaced by a different amino acid. Advantageously, theasparagine at amino acid position 297 is substituted with anon-glutamine, non-aspartic acid residue (e.g., a non-negatively chargedamino acid, any conservative substitution, an amino acid with apositively charged side chain, an amino acid with a polar uncharged sidechain, an amino acid with a hydrophobic side chain, e.g. a serine). Theantibody will thus have a constant region with a N297X substitution (aN297X variant antibody), wherein X is any amino acid other thanasparagine, glutamine or aspartic acid.

In another example, an antibody having a glutamine naturally present atheavy chain residue 295 and an asparagine at residue 297 (EU numbering)can comprise a modification at residues 295 and 297 such that theglutamine at residue 295 is deleted or replaced by a different aminoacid (e.g., a non-negatively charged amino acid and the asparagine atresidue 297 is replaced by a glutamine which then serves as the acceptorglutamine. The antibody will thus have a constant region with Q295X andN297Q substitutions (a Q295X N297Q variant antibody), wherein X is anyamino acid other than glutamine, optionally wherein the substitution isa non-negatively charged amino acid.

In another example, an antibody having an acceptor glutamine (e.g. aglutamine naturally present at heavy chain residue 295) and anasparagine at residue 297 (EU numbering) comprises a modification at anon-297 residue (a residue that is not at position 297, EU numbering) inan Fc domain (e.g. CH1, CH2 and/or CH3 domain), wherein the modificationabrogates N297-linked glycosylation. Such an antibody will have anacceptor glutamine (e.g. at residue 295) together with an aglycosylatedasparagine at residue 297. For example, modifications leading toelimination of asparagine-linked glycosylation at N297 include asubstitution at residue T299 (or optionally additional substitutions atother residues, e.g. substitutions at both T299 and S298), see, e.g.,any of the mutations and combinations of mutations disclosed in Sazinskyet al. 2008 Proc. Nat. Acad. Sci. U.S.A. 105(51):20167-20172. Anexemplary antibody can thus have a constant region with a T299Xsubstitution (a T299X variant antibody), wherein X is an amino acidother than threonine, wherein the modification abrogates N297-linkedglycosylation.

In one embodiment, an antibody comprises a heavy chain constant regioncomprising an amino acid sequence HNAKTKPREEQ-X¹—X²—STYRVVSVLT (SEQ IDNO: 3), wherein X′ is Y (tyrosine) or F (phenylalanine) and X² is anamino acid other than D (aspartic acid). Optionally, X² is anon-negatively charged amino acid, any conservative substitution, anamino acid with a positively charged side chain, an amino acid with apolar uncharged side chain, an amino acid with a hydrophobic side chain,e.g. a serine. In one embodiment, an antibody comprises a heavy chainconstant region comprising an amino acid sequenceHNAKTKPREEQ-X¹—NS—X²—YRVVSVLT (SEQ ID NO: 4), wherein X′ is Y or F andX² is an amino acid other than T (threonine).

In one embodiment, an antibody comprises a heavy chain constant regionamino acid sequence of SEQ ID NOS: 6 or 8, a fragment of at least 10,25, 50, 100 amino acid residues thereof, or an amino acid sequence atleast 80%, 90%, 95% or 99% identical to the foregoing, wherein the heavychain constant region comprises a glutamine residue flanked at the +2position by a non-aspartic acid residue. Optionally the residue at theat the +2 position is an amino acid other than an asparagine, glutamineor aspartic acid. Optionally the residue at at the +2 position is anon-negatively charged amino acid. Optionally, the heavy chain constantregion comprises an acceptor glutamine at residue 174 of SEQ ID NOS: 6or 8, corresponding to EU numbering residue 295, and a non-aspartic acidresidue at residue 176 of SEQ ID NOS: 6 or 8, corresponding to EUresidue 297.

Such antibodies lacking aspartic acid at the +2 position will form asubstrate for efficient TGase mediated conjugation of linkers comprisinglarge and/or hydrophobic moieties.

Engineered antibodies can be prepared by a variety of methods whichinclude, but are not limited to, isolation from a natural source (in thecase of naturally occurring amino acid sequence variants), preparationby site-directed (or oligonucleotide-mediated) mutagenesis (Carter(1985) et al Nucleic Acids Res. 13:4431-4443; Ho et al (1989) Gene(Amst.) 77:51-59; Kunkel et al (1987) Proc. Natl. Acad. Sci. USA 82:488;Liu et al (1998) J. Biol. Chem. 273:20252-20260), PCR mutagenesis (Itoet al (1991) Gene 102:67-70; and Vallette et al (1989) Nuc. Acids Res.17:723-733) and cassette mutagenesis (Wells et al (1985) Gene34:315-323) of an earlier prepared DNA encoding the polypeptide.Mutagenesis protocols, kits, and reagents are commercially available,e.g. QuikChange® Multi Site-Direct Mutagenesis Kit (Stratagene, LaJolla, Calif.). Single mutations are also generated by oligonucleotidedirected mutagenesis using double stranded plasmid DNA as template byPCR based mutagenesis (Sambrook and Russel, (2001) Molecular Cloning: ALaboratory Manual, 3rd edition). Variants of recombinant antibodies maybe constructed also by restriction fragment manipulation or by overlapextension PCR with synthetic oligonucleotides. Mutagenic primers encodethe cysteine codon replacement(s). Standard mutagenesis techniques canbe employed to generate DNA encoding such mutant cysteine engineeredantibodies (Sambrook et al Molecular Cloning, A Laboratory Manual. ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; andAusubel et al Current Protocols in Molecular Biology, Greene Publishingand Wiley-Interscience, New York. N.Y., 1993).

Antibodies may be chemically synthesized using known oligopeptidesynthesis methodology or may be prepared and purified using recombinanttechnology. In vitro protein synthesis may be performed using manualtechniques or by automation.

In one embodiment of the invention provides a method of preparing(making) an antibody for use in a one-step TGase-mediated couplingreaction (e.g. with a linker of Formula Ia, Ib or Ic), comprising:

-   -   (a) providing a parent antibody having in a heavy chain: (i) an        acceptor glutamine residue at residue 295 and (ii) an asparagine        at position 297; and    -   (b) substituting the asparagine present at position 297 of said        parent antibody by a non-glutamine residue, in order to generate        an antibody having an acceptor glutamine residue at residue 295        and lacking N-linked glycosylation at position 297. Optionally,        the amino acid that replaces the asparagine at position 297 is a        residue other than an aspartic acid.

In one embodiment of the invention provides a method of preparing(making) an antibody for use in a one-step TGase-mediated couplingreaction (e.g. with a linker of Formula Ia, Ib or Ic), comprising:

-   -   (a) providing a parent antibody having in a heavy chain: (i) an        acceptor glutamine residue at residue 295 and (ii) an threonine        at position 299; and    -   (b) modifying (e.g. substituting with another amino acid) the        threonine at position 299 of said parent antibody in order to        generate an antibody having an acceptor glutamine residue at        residue 295 and lacking N-linked glycosylation at position 297.        In one embodiment of the invention provides a method of        preparing (making) an antibody for use in a one-step        TGase-mediated coupling reaction (e.g. with a linker of Formula        Ia, Ib or Ic), comprising:    -   (a) providing a parent antibody having in a heavy chain: (i) a        glutamine residue at residue 295 and (ii) an asparagine at        position 297; and    -   (b) substituting the glutamine present at position 295 of said        parent antibody by any amino acid (e.g. other than        non-glutamine, a conservative substitution, a non-aspartic acid        residue), and substituting the asparagine at position 297 by a        glutamine, in order to generate an antibody having an acceptor        glutamine residue and lacking N-linked glycosylation at residue        297, and lacking an acceptor glutamine at position 295.

The method may optionally further comprises a step (c) reacting theantibody of step (b) with a compound of Formula I (e.g. a compound ofFormula Ia, Ib or Ic) in the presence of a TGase under suitableconditions, such that an antibody of Formula II, IVa or IVb is formed.

In one embodiment of the invention provides a method of preparing(making) a glutamine engineered antibody, comprising:

-   -   (a) introducing one or more acceptor glutamine residues into a        parent antibody in order to generate a glutamine engineered        antibody; and    -   (b) reacting the glutamine engineered antibody with a compound        of Formula I (e.g. a compound of Formula Ia, Ib or Ic) in the        presence of a TGase under suitable conditions, such that an        antibody of Formula II, IVa or IVb is formed.

Step (a) of the method of preparing a glutamine engineered antibody maycomprise:

-   -   (i) mutagenizing a nucleic acid sequence encoding the glutamine        engineered antibody;    -   (ii) expressing the glutamine engineered antibody; and    -   (iii) isolating and purifying the glutamine engineered antibody.

In one example, the cancer antigen is human L1-CAM (CD171; L1 celladhesion molecule) which has been found to be expressed in a variety ofcancers (see, e.g., Kajiwara et al, (2011) Am. J. Clin. Pathol. 136 (1),138-144). The L1-CAM nucleotide and amino acid sequences are disclosedin Genbank accession numbers NM_(—)024003.2 and NP_(—)076493.1,respectively, the disclosures of which are incorporated by reference. Anexample of an anti-L1-CAM antibody suitable for use in accordance withthe invention is a chCE7-derived antibody, e.g, having a heavy chaincomprising CDRs (e.g., CDR-H1, —H2 and —H3) from chCE7 heavy chain shownin SEQ ID NO: 1 and a light chain comprising CDRs (e.g., CDR-L1, -L2 and-L3) from chCE7 heavy chain shown in SEQ ID NO 2, optionally wherein anyof said CDRs further comprises one, two, three, four or five amino acidmodifications so long as the antibody retains specific binding toL1-CAM. ChCE7 is composed of murine VL and murine VH fused to the Fcpart of human IgG1 (see, e.g., Jeger et al., (2010) Angew. Chem. Int.,49, 9995-9997). chCE7 optionally comprises specific mutations wereintroduced in the CH2 domain of the chCE7 heavy chain using overlappingpolymerase chain reaction (PCR) and standard molecular biologytechniques (Q295N and N297Q variants), including chCE7 N297Q variantswith an acceptor glutamine at position 295 and 297, andchCE7aglQ295N,N297Q variants with an acceptor glutamine at position 297.

An exemplary humanized CE7 antibody comprises a VH domain comprising aCDR-H1 sequence corresponding to residues 31-35 of SEQ ID NO: 1, aCDR-H2 sequence corresponding to residues 50-66 of SEQ ID NO: 1, and aCDR-H3 sequence corresponding to residues 99-109 of SEQ ID NO:1, whereinany CDR may optionally comprise one, two, three, four or more amino acidsubstitutions. An exemplary humanized CE7 antibody may also oralternatively comprise a VL domain comprising a CDR-L1 sequencecorresponding to residues 24-34 of SEQ ID NO: 2, a CDR-L2 sequencecorresponding to residues 50-56 of SEQ ID NO: 2, and an CDR-L3 sequencecorresponding to residues 89-95 (or 89-97) of SEQ ID NO: 2, wherein anyCDR may optionally comprise one, two, three, four or more amino acidsubstitutions.

Fragments and derivatives of antibodies of this invention (which areencompassed by the term “antibody” or “antibodies” as used in thisapplication, unless otherwise stated or clearly contradicted bycontext), can be produced by techniques that are known in the art.“Fragments” comprise a portion of the intact antibody, generally theantigen binding site or variable region. Examples of antibody fragmentsinclude Fab, Fab′, Fab′-SH, F(ab′)2, and Fv fragments; diabodies; anyantibody fragment that is a polypeptide having a primary structureconsisting of one uninterrupted sequence of contiguous amino acidresidues (referred to herein as a “single-chain antibody fragment” or“single chain polypeptide”), including without limitation (1)single-chain Fv molecules (2) single chain polypeptides containing onlyone light chain variable domain, or a fragment thereof that contains thethree CDRs of the light chain variable domain, without an associatedheavy chain moiety and (3) single chain polypeptides containing only oneheavy chain variable region, or a fragment thereof containing the threeCDRs of the heavy chain variable region, without an associated lightchain moiety; and multispecific antibodies formed from antibodyfragments. Included, inter alia, are a nanobody, domain antibody, singledomain antibody or a “dAb”.

The DNA of a hybridoma producing an antibody of the invention may bemodified so as to encode a fragment of the invention. The modified DNAis then inserted into an expression vector and used to transform ortransfect an appropriate cell, which then expresses the desiredfragment.

The fragment will comprise a variable region domain that will generallybe covalently attached to at least one, two or more glutamine residuecovalently linked through a —NH—(C)_(n)—X-L moiety (and optionallyfurther a V and/or Y moiety, optionally further an R or RR′ moiety, to amoiety-of-interest Z, e.g. a polymer molecule, a drug, a radioactivemoiety. The variable region will comprise hypervariable region or CDRsequences, and FR sequences.

The location of the glutamine residue may be varied according to thesize and nature of the antibody fragment required. Thus, in one extremeexample an acceptor glutamine residue to be conjugated to a lysine-basedlinker of Formula I may be attached directly to a C-terminal amino acidof the variable region domain. This may be for example the C-terminus ofa VH or VL chain as described above. If desired, in this example,further amino acids, including further acceptor glutamine residues, maybe covalently linked to the C-terminus of the first glutamine residue.In one example, a peptide “tag” comprising one or more non-glutamineresidues followed by an acceptor glutamine residue (the acceptorglutamine residue is C-terminal to the non-glutamine residue in the tag)is attached directly to a C-terminal amino acid of the variable regiondomain. In one example, a peptide “tag” comprising one or more glutamineresidues followed by one or more non-glutamine residues (thenon-glutamine residues are C-terminal to the glutamine residue in thetag) is attached directly to a C-terminal amino acid of the variableregion domain. A peptide tag can be of any suitable length, e.g a tagmay comprise between 2 and 50, preferably 2 and 20 or 2 and 10 aminoacid residues.

In practice however, it is generally preferable that the variable regiondomain is covalently attached at a C-terminal amino acid to at least oneother antibody domain or a fragment thereof which contains, or isattached to one or more acceptor glutamine residues. Thus, for examplewhere a VH domain is present in the variable region domain this may belinked to an immunoglobulin CH1 domain or a fragment thereof. Similarlya VL domain may be linked to a CK domain or a fragment thereof. In thisway for example the fragment according to the invention may be a Fabfragment wherein the antigen binding domain contains associated VH andVL domains covalently linked at their C-termini to a CH1 and CK domainrespectively. The CH1 domain may be extended with further amino acids,for example to provide a hinge region domain as found in a Fab′fragment, or to provide further domains, such as antibody CH2 and CH3domains. In one example, a polypeptide “tag” comprising one or aplurality (e.g. 2, 3, 4, 5, 6) non-glutamine residues followed by aglutamine residue (the glutamine residue is C-terminal to thenon-glutamine residue in the tag) is attached directly to a C-terminalamino acid of a full or truncated CH1, CH2 or CH3 domain, or to aC-terminal amino acid of a full or truncated CK domain. In one example,a polypeptide “tag” comprising one or more glutamine residues followedby one or more non-glutamine residues (the non-glutamine residues areC-terminal to the glutamine residue in the tag) is attached directly toa C-terminal amino acid of a full or truncated CH1, CH2 or CH3 domain,or to a C-terminal amino acid of a full or truncated CK domain.

The present invention provides an antibody fragment in which thevariable region domain is monomeric and comprises an immunoglobulinheavy (VH) or light (VL) chain variable domain, or is dimeric andcontains VH—VH, VH-VL or VL-VL dimers in which the VH and VL chains arenon-covalently associated or covalently coupled, wherein the fragment(i.e. the VL and/or VH) is covalently linked through a —NH—(C)_(n)—X-Lmoiety (and optionally further a V and/or Y moiety, optionally furtherL′, V′, Y′, and (RR′)moieties, to a moiety-of-interest Z, e.g. a polymermolecule, a drug, a radioactive moiety. Preferably each VH and/or VLdomain is covalently attached at a C-terminal amino acid to at least oneother antibody domain or a fragment thereof.

In one embodiment, the invention provides a monovalent antibody fragmentcomprising a heavy chain and a light chain, wherein: said heavy chainconsists of a VH domain covalently linked at its C-terminus to a CH1domain; said light chain consists of a VL domain, which is complementaryto the VH domain, covalently linked at its C-terminus to a CL domain;said CH1 domain comprises (e.g., the CH₁ is extended) to provide a hingedomain which comprises a glutamine residue; and the glutamine residue inthe hinge domain is covalently linked through a —NH—(C)_(n)—X-L moiety.In another embodiment, the invention provides a monovalent antibodyfragment comprising a heavy chain and a light chain, wherein: said heavychain consists of a VH domain covalently linked at its C-terminus to aCH1 domain; said light chain consists of a VL domain, which iscomplementary to the VH domain, covalently linked at its C-terminus to aCL domain; said CL domain comprises (e.g., the CL is extended) toprovide a hinge domain which comprises a glutamine residue; and theglutamine residue in the hinge domain is covalently linked through a—NH—(C)_(n)—X-L moiety.

The invention has the advantage over cysteine-based conjugation methodsof not requiring cysteine-engineering to remove cysteine residues thatcould react with the moiety of interest. Thus, in one embodiment, theantibody fragment of the invention contains cysteine residues in the VH,CH1, VL and CL domains that are in disulphide linkage to each other;preferably the antibody fragment comprises some or all cysteines capableof forming interchain disulfide bonds in naturally present in VH, CH1,VL and CL domains. In one embodiment, the antibody comprises a lightchain comprising a cysteine at position 214 and/or a heavy chaincomprising a cysteine at position 127, 128, 233 and/or 235.

In one embodiment, the antibody fragment is linked through a—NH—(C)_(n)—X-L moiety to a polymer (e.g. a PEG-comprising molecule).

Lysine-Based Linkers

Certain aspects of the invention are directed to a linking reagent thatcan be attached, by the action of a TGase, to a polypeptide at aglutamine residue (Q) within the sequence of the polypeptide, forexample an antibody (Ab). The linking reagent contains a primary aminethat functions as a TGase substrate for conjugation onto an acceptorglutamine on an antibody, for example a lysine derivative (Lys),including but not limited to a lysine amino acid residue, or afunctional equivalent thereof, that is connected to at least onereactive group (R). In one embodiment, a moiety-of-interest (Z), andoptionally one or more other groups, are attached to the linkingreagent. In one embodiment, for use in a multi-step conjugation process,a plurality of reactive groups, preferably non-complementary reactivegroups, can be attached to the linking reagent. The reactive group ispreferably a functionality that is insensitive to water but selectivelyundergoes a very high conversion addition reaction with a complementaryreagent.

The lysine derivative can be a 2 to 20 alkyl or heteroalkyl chain, or afunctional equivalent thereof, with an H₂N, H₂NOCH₂, H₂NCH₂(aminomethylene) group or a protected H₂N, H₂NOCH₂, H₂NCH₂ grouppositioned at one or more ends of the alkyl or heteroalkyl chain. Theheteroalkyl chain can be a chain of 3 to 20 atoms where one or morenon-terminal atoms can be other than carbon, for example oxygen, sulfur,nitrogen, or other atoms. The oxygen, sulfur, or nitrogen atom can be ofan ether, ester, thioether, thioester, amino, alkylamino, amido oralkylamido functionality within the carbon chain.

The heteroalkyl chain can be an oligo (ethylene oxide) chain. Thefunctionality within the alkyl or heteroalkyl chain can be included tocouple the reactive group to the H₂N, H₂NOCH₂, H₂NCH₂ group or protectedH₂N, H₂NOCH₂, H₂NCH₂ group. The alkyl or heteroalkyl chain can besubstituted or unsubstituted. The substituents can be alkyl groups, arylgroups, alkyl aryl groups, carboxylic acid groups, amide groups, hydroxygroups, or any other groups that do not compete with the amino groupfor, or inhibit, conjugation with a glutamine residue of the protein.Typically, when a substituent is present, its presence is in aconvenient starting material, such as the carboxylic acid group oflysine, from which the lysine derivative results. The H₂N, H₂NOCH₂,H₂NCH₂ end of a alkyl or heteroalkyl chain is necessarily included inthe linking reagent.

Exemplary starting materials for the functional equivalent of lysine canbe an α,ω-diaminoalkane, for example, 1,2-diaminoethane,1,3-diaminopropane, 1,4-diaminobutane, 1,5-diaminopentane,1,6-diaminohexane, 1,7-diaminoheptane, 1,8-diaminooctane,1,9-diaminononane, 1,10-diaminodecane, 1,11-diaminoundecane, or1,12-diaminododecane. Other starting materials for the functionalequivalent of a lysine derivative can be a,w-diamino oligo (ethyleneoxide), for example, H₂N(CH₂CH₂O)_(x)CH₂CH₂NH₂ where x is 1 to about 6.The α,ω-diamino oligo (ethylene oxide) can be a single oligomer or itcan be a mixture of oligomers where x defines an average size. Anexemplary protected H₂NCH₂ is the tert-butylcarbamate protected amine oftert-butyl N-(5-aminopentyl)carbamate (N-Boc-cadaverin).

The linking reagent, a pharmaceutically acceptable salt or solvatethereof, or a protein conjugated linking reagent may comprise thegeneral Formula Ia or Ib. Formulae Ia (having an Z group) and Ib (havinga R group) are shown as follows:

G-NH—(C)_(n)—X-L-(V—(Y—(Z)_(z))_(q))_(r)  Formula Ia;

G-NH—(C)_(n)—X-L-(V—(Y—(R)_(z))_(q))_(r)  Formula Ib

or a pharmaceutically acceptable salt or solvate thereof

wherein:

G is an H, amine protecting group, or an immunoglobulin (Ab) or otherprotein attached via an amide bond;

(C)_(n) is a substituted or unsubstituted alkyl or heteroalkyl chain,optionally where the carbon adjacent to the nitrogen is unsubstituted,optionally wherein any carbon of the chain is substituted alkoxy,hydroxyl, alkylcarbonyloxy, alkyl-S—, thiol, alkyl-C(O)S—, amine,alkylamine, amide, or alkylamide (e.g. with a O, N or S atom of anether, ester, thioether, thioester, amine, alkylamine, amide, oralkylamide);

n is an integer selected from among the range of 2 to 20, preferably 3to 6;

X is NH, O, S, or absent;

L is a bond or a carbon comprising framework of 1 to 200 atomssubstituted at one or more atoms, optionally wherein the carboncomprising framework is a linear hydrocarbon, a symmetrically orasymmetrically branched hydrocarbon, monosaccharide, disaccharide,linear or branched oligosaccharide (asymmetrically branched orsymmetrically branched), an amino acid, a di-, tri-, tetra-, oroligopeptide, other natural linear or branched oligomers (asymmetricallybranched or symmetrically branched), or a dimer, trimer, or higheroligomer (linear, asymmetrically branched or symmetrically branched)resulting from any chain-growth or step-growth polymerization process;

r is an integer selected from among 1, 2, 3 or 4;

q is an integer selected from among 1, 2, 3 or 4; and

z is an integer selected from among 1, 2, 3 or 4;

V is independently absent, a bond or a continuation of a bond if L is abond, a non-cleavable moiety or a conditionally-cleavable moiety,optionally following prior conditional transformation, which can becleaved or transformed by a chemical, photochemical, physical,biological, or enzymatic process (e.g. cleavage of V ultimately leadingto release of one or more moieties subsequently or ultimately linked toV, for example a Z moiety). In some embodiments, V is, preferably, adi-, tri-, tetra-, or oligopeptide as described below in the sectionentitled “The V Moiety”;

Y is independently absent, a bond or a continuation of a bond if V is abond or continuation of a bond, or a spacer system (e.g., aself-eliminating spacer system or a non-self-elimination spacer system)which is comprised of 1 or more spacers;

Z is a moiety that improves the pharmacokinetic properties, atherapeutic moiety or a diagnostic moiety; and

R is a reactive moiety, preferably a moiety comprising an unprotected orprotected thiol, maleimide, haloacetamide, o-phoshenearomatic ester,azide, fulminate, alkyne, cyanide, anthracene, 1,2,4,5-tetrazine,norbornene, other stained or otherwise electronically activated alkeneor, optionally, a protected or unprotected amine when X is absent and L,V, or Y is other than a bond or a continuation of a bond. In analternative embodiment R is a reactive moiety, preferably a moietycomprising an unprotected or protected thiol, an unprotected orprotected amine, maleimide, haloacetamide, o-phoshenearomatic ester,azide, fulminate, alkyne, cyanide, anthracene, 1,2,4,5-tetrazine,norbornene, other stained or otherwise electronically activated alkene,provided that R is not an amine when n=5 and X, L, V and Y are absent.Optionally, R is not an amine when n=4 and X, L, V and Y are absent.When more than one R group is present in a compound of the formula Ib,the R groups will preferably be compatible such that no R group is acomplementary reagent to any other R group.

The (C)_(n) group may for example be a straight, branched and/or cyclicC₂₋₃₀ alkyl, C₂₋₃₀ alkenyl, C₂₋₃₀ alkynyl, C₂₋₃₀ heteroalkyl, C₂₋₃₀heteroalkenyl, C₂₋₃₀ heteroalkynyl, optionally wherein one or morehomocyclic aromatic compound radical or heterocyclic compound radicalmay be inserted; notably, any straight or branched C₂₋₅ alkyl, C₅₋₁₀alkyl, C₁₁₋₂₀ alkyl, —O—C₁₋₅ alkyl, —O—C₅₋₁₀ alkyl, —O—C₁₁₂₀ alkyl,CH₂—(CH₂—O—CH₂)₁₋₁₂—CH₂ or (CH₂—CH₂—O—)₁₋₁₂, an amino acid, anoligopeptide, glycan, sulfate, phosphate or carboxylate.

In one example the (C)_(n) group is a carbon comprising frameworksubstituted with one or more O atoms. In one embodiment, the carbonadjacent to the nitrogen is substituted with an O atom. In oneembodiment, the carbon adjacent to the nitrogen is unsubstituted. In oneembodiment, the (C)_(n) group is or comprises an ethylene oxide group,e.g. a CH₂—(CH₂—O—CH₂)_(n)—CH₂ group or an (CH₂—CH₂—O—)_(n), where n isan integer from 1 to 10.

The L group can be a carbon comprising framework, where L is a linearhydrocarbon, a symmetrically or asymmetrically branched hydrocarbon,monosaccharide, disaccharide, linear or branched oligosaccharide(asymmetrically branched or symmetrically branched), an amino acid, adi-, tri-, tetra-, or oligopeptide, other natural oligomer, dimer,timer, or higher oligomer (linear asymmetrically branched orsymmetrically branched) resulting from any chain-growth or step-growthpolymerization process. For example, L may comprise or be a straight,branched and/or cyclic C₂₋₃₀ alkyl, C₂₋₃₀ alkenyl, C₂₋₃₀ alkynyl, C₂₋₃₀heteroalkyl, C₂₋₃₀ heteroalkenyl, C₂₋₃₀ heteroalkynyl, optionallywherein one or more homocyclic aromatic compound radical or heterocycliccompound radical may be inserted; notably, any straight or branched C₂₋₅alkyl, C₅₋₁₀ alkyl, C₁₁₋₂₀ alkyl, —O—C₁₋₅ alkyl, —O—C₅₋₁₀ alkyl,—O—C₁₁₋₂₀ alkyl, CH₂—(CH₂—O—CH₂)₁₋₃₀—CH₂ or (CH₂—CH₂)₁₋₃₀, e.g.,(CH₂—CH₂—O—)₁₂, (CH₂—CH₂—O—)₁₋₂₄,an amino acid, an oligopeptide, glycan,sulfate, phosphate, carboxylate. Optionally, L is absent.

L, V and/or Y have r, q, and/or z sites of attachment for the respectiveV, Y, and Z or R groups, where r and q represent the degree of branchingor polymerization. The sites of attachment can comprise a bond orcomprise a functional group selected from an alkene, alkyne, ether,thioether, ester, thioester, amine, amide, alkylamide, or otherfunctional group readily generated by a condensation or additionreaction.

In one example the carbon comprising framework of the L group isoptionally substituted with one or more O atoms. In one embodiment, theL group comprises one or more ethylene oxide groups (CH₂—O—CH₂).Optionally, the L group comprises a carbon framework comprising a(CH₂—CH₂—O—)_(n) group, wherein n is an integer selected among the rangeof 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10).

In Formulae Ia, Ib, II, IVa and IVb, the linking group L links theaminopeptidyl moiety —NH—(C)_(n)—X to the reactive group R or Z,optionally through one or more V and/or Y moieties where present. L maybe a bond connecting V, Y, R or Z directly to the aminopeptidyl moiety.In another aspect, however, L is a linking group that functionally linksor spaces the one or more moieties V and/or Y reactive moiety R ormoiety of interest (Z). In Formulae Ib, Ic, II and IVb, spacing improvesefficiency and completion of BTGase coupling, make additionally thereactive moiety R more accessible to the reaction partner, for examplewhen the reactive moiety is present on a lysine-based linker and coupledto the antibody and then brought into contact with a reaction partner.In Formulae Ia and IVa, the linking group L links the aminopeptidylmoiety —NH—(C)_(n)—X to the moiey-of-interest (Z), optionally throughone or more V and/or Y moieties where present. L may be a bondconnecting V, Y or Z directly to the aminopeptidyl moiety. In anotheraspect, however, L is a linking group that functionally links or spacesthe one or more moieties V and/or Y reactive moiety Z. In Formulae Iaand IVa, spacing improves efficiency and completion of BTGase coupling,providing for highly homogenous compounds. In antibodies comprising afunctionalized acceptor glutamine of Formula IVa or IVb spacing may alsoprovide for a better accessibility of V, which in the case of enzymaticcleavage or transformation of V, may improve the rate at which V istransformed and/or cleaved.

L and (C)_(n) groups can be configured based on the overall structure ofthe linker that is to be used. Particularly when a multi-step method ofthe invention is used and the linker (e.g. the linker of Formula Ia, Ibor Ic is free of or does not comprise a large, charged or hydrophobicmoiety (e.g. a cyclic, polycyclic or macrocyclic moiety), the L groupmay be a bond or a shorter carbon framework. For example, L mayrepresent or comprise a carbon framework of 1, 2, 3, 4, 5, or 6 linearcarbon atoms, unsubstituted or optionally substituted at one or moreatoms. Preferably, where L additionally comprises other groups, the 5-20linear carbon atoms will be adjacent to the (C)_(n) group, or wherepresent, the X group.

When a linker (e.g. the linker of Formula Ia, Ib or Ic or an antibody ofFormula II, IVa or IVb) comprises a large, charged or hydrophobic moiety(e.g. a cyclic, polycyclic or macrocyclic moiety), for example, whereinV, Y and/or Z comprises a large, charged or hydrophobic moiety (e.g. acyclic, polycyclic or macrocyclic moiety), the L group may be longercarbon framework. For example, L may represent or comprise a carbonframework of:

a) 2-30 linear carbon atoms optionally substituted at one or more atoms;

b) 2-15 linear carbon atoms optionally substituted at one or more atoms;

c) 5-20 linear carbon atoms optionally substituted at one or more atoms;

d) 5-30 linear carbon atoms optionally substituted at one or more atoms;

e) 5-15 linear carbon atoms optionally substituted at one or more atoms;or

f) 4, 5 or 6 linear carbon atoms optionally substituted at one or moreatoms.

Preferably, the 5-20 linear carbon atoms will be adjacent to (thecontinuation of) the (C)_(n) group, or where present, the X group.

In some embodiments, L is a —(C═O)—C₁₋₆ alkyl group. In someembodiments, L is a C₁₋₆ alkoxy-C₁₋₆ alkyl group. In some embodiments, Lis a —(C═O)—C₁₋₆ alkoxy-C₁₋₆ alkyl group. In some embodiments, L is a—(C═O)—C₁₀₋₂₀ alkyl group. In some embodiments, L is a C₁₋₆ alkyl group.In some embodiments, L is a C₁₀₋₂₀ alkyl group. In some embodiments, Lis a —(C═O)—O—C₁₋₆ alkyl group. In some embodiments, L is a—(C═O)—O—C₂₋₂₀ alkyl group. In some embodiments, L is a —(C═O)— group.In some embodiments, L is selected from among

In some embodiments, L is or comprises an amino acid or a di-,tri-tetra- or oligopeptide. In some embodiments, L is selected fromamong alanine, arginine, asparagine, aspartic acid, cysteine, glutamine,glutamic acid, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, serine, threonine, tryptophan,tyrosine, valine, and citrulline

In any of the compounds of the invention (e.g. in any of Formula I, IIand/or IV), linking element (L) can optionally be characterized ashaving a chain length of at least 2.8 Angstroms, 3, Angstroms, 4Angstroms, 5 Angstroms, 10 Angstroms, 15 Angstroms, 18 Angstroms, 30Angstroms, 40 Angstroms or 60 Angstroms. Optionally L has a length of nomore than 100 Angstroms, optionally no more than 60 AngstromsOptionally, L is characterized as having a length of between 2.8, 3, 4,5, 10, 20 or 30 Angstroms and 60 Angstroms. Optionally, L ischaracterized as having a length of between 2.8 and 19 Angstroms, orbetween 4 and 19 Angstroms.

Examples of compounds of Formula Ia include but are not limited tocompound having the (C)_(n), X, L, V, Y and Z groups shows in Table 2herein. Examples of compounds of Formula Ib include but are not limitedto compound having the (C)_(n), X, L, V, Y and R groups shows in Table 3herein. R groups in Table 3 indicated as (S) can also be S(C═O)CH₃ whenpresent as a protected reactive group. The symbol (−) in the tablesindicates that the particular X, L, V or Y moiety is absent. V and Ygroups, for example, can comprise any structural features in thesections titled “The V Moiety” and “The Y Moiety” herein. The L, Vand/or Y groups of Formulae Ia and Ib represented in each of Tables 2and 3 can have r, q, and/or z sites of attachment for the respective V,Y, and R or Z groups, where r and q represent the degree of branching orpolymerization; r, q, and/or z can be selected from 1, 2, 3 or 4.

A compound of this invention may contain more than one L moiety. Any L′moiety can be defined in the same way as a L moiety. The L moieties mayor may not be the same. The linking group L may be a water-solublemoiety or contain one or more water-soluble moieties, such that Lcontributes to the water solubility of a compound of Formula (I)-(VI).An L may also be a moiety or contain one or more moieties that reduce(s)aggregation, which may or may not be a moiety/moieties that alsoincrease(s) the water solubility.

L may be for example a linear linker or a branched linker. In oneaspect, the L moiety is branched, optionally further a dendriticstructure, so that it can be connected to at least two, three, four ormore V, Y or R moieties (or Z where applicable). Each V—Y moiety ishowever only attached once to an L moiety. Branching can occur at one ormore branching atoms that may for example be carbon, nitrogen, silicon,or phosphorus.

When the lysine-based linker comprises branching in L, the number ofbranches in L that are connected to V and/or Y will generally beprepared so as to equal the total number of branches available forreaction. That is, in preparing the lysine-based linker, chemicalconversion will preferably be carried to completion, thereby maintainthe controlled stoichiometry offered by the site-specific TGase-mediatedconjugation approach. Thus, preferably, when L is branched, compounds ofthis invention will be functionalized such that each L, V or Y isconnected to a R or Z moiety, such that the components of the mixture ofantibodies (or the lysine-based linker during preparation) substantiallyall have the same r value. For example, it can be specified that 90%,95%, 98% of the antibodies or the lysine-based linker have the same rvalue. In one embodiment, L is a linear linker. In another embodiment, Lis a branched linker.

Any one of the L moieties disclosed herein can be utilized in FormulaIa, Ib, Ic, II, IVa, and IVb. Any one of the L moieties described hereincan be used in combination with any of the (C)_(n), X, V, Y, Z, R, M, z,q, and r groups described herein. Any one of the L′ moieties disclosedherein can be utilized in Formula III. Any one of the L′ moietiesdescribed herein can be used in combination with any of the R′, V′, Y′,Z, z′, q′, and r′ groups described herein.

Exemplary linkers of Formula Ia include but are not limited to:

Exemplary linkers of Formula Ib include but are not limited to:

The Reactive Moiety R

R is a reactive moiety, for example a moiety comprising an unprotectedor protected bioorthogonal-reaction compatible reactive group, forexample an unprotected or protected thiol, epoxide, maleimide,haloacetamide, o-phoshenearomatic ester, azide, fulminate, sulfonateester, alkyne, cyanide, amino-thiol, carbonyl, aldehyde, generally anygroup capable of oxime and hydrazine formation, 1,2,4,5-tetrazine,norbornene, other stained or otherwise electronically activated alkene,a substituted or unsubstituted cycloalkyne, generally any reactivegroups which form via bioorthogonal cycloaddition reaction a 1,3- or1,5-disubstituted triazole, any diene or strained alkene dienophile thatcan react via inverse electron demand Diels-Alder reaction, a protectedor unprotected amine, a carboxylic acid, an aldehyde, or an oxyamine.

When more than one R group is present in a compound of the formula, theR groups will preferably be compatible such that no R group is acomplementary reagent to any other R group. The L, V and/or Y groups offormulae I-IV can have r, q, and/or z sites of attachment for therespective V, Y, and R groups, where r and q represent the degree ofbranching or polymerization. The sites of attachment can comprise a bondor comprise a functional group selected from an alkene, alkyne, ether,thioether, ester, thioester, amine, amide, alkylamide, or otherfunctional group readily generated by a condensation or additionreaction.

The reactive group of the linking reagent can for example chosen toundergo thio-maleimide (or haloacetamide) addition, Staudinger ligation,Huisgen 1,3-cycloaddition (click reaction), or Diels-Alder cycloadditionwith a complementary reactive group attached to an agent comprising atherapeutic moiety, a diagnostic moiety, or any other moiety for adesired function.

Optionally, two or more compatible reactive groups can be attached tothe linking reagent.

In one embodiment, the reactive group is a haloacetamide, (e.g.bromo-acetamide, iodo-acetamide, cloro-acetamide). Such reactive groupswill be more stable in vivo (and in serum) compared with maleimidegroups.

In one embodiment, the reactive group is a reagent capable of undergoinga “click” reaction. For example a 1,3-dipole-functional compound canreact with an alkyne in a cyclization reaction to form a heterocycliccompound, preferably in the substantial absence of added catalyst (e.g.,Cu(I)). A variety compounds having at least one 1,3-dipole groupattached thereto (having a three-atom pi-electron system containing 4electrons delocalized over the three atoms) can be used to react withthe alkynes disclosed herein. Exemplary 1,3-dipole groups include, butare not limited to, azides, nitrile oxides, nitrones, azoxy groups, andacyl diazo groups.

Examples include o-phosphenearomatic ester, an azide, a fulminate, analkyne (including any strained cycloalkyne), a cyanide, an anthracene, a1,2,4,5-tetrazine, or a norbornene (or other strained cycloalkene).

In one embodiment, R is a moiety having a terminal alkyne or azide; suchmoieties are described for example in U.S. Pat. No. 7,763,736, thedisclosure of which is incorporated herein by reference. Suitablereaction conditions for use of copper (and other metal salt) ascatalysts of click-reactions between terminal alkynes and azides areprovided in U.S. Pat. No. 7,763,736.

In one embodiment, R is a substituted or unsubstituted cycloalkyne.Cycloalkynes, including heterocyclic compounds, will preferably be usedin linking reagents of the invention in which an L group is present,preferably wherein L is an alkyl or heteroalkyl chain of 3-30,optionally 5-30 or 5-15 linear carbon atoms, optionally substituted atone or more atoms. Optionally, L is a (CH₂—CH₂—O)₁₋₂₄ group or a(CH₂)_(x1-)(CH₂—O—CH₂)₁₋₂₄—(CH₂)_(x2-), wherein x1 and x2 areindependently an integer selected among the range of 0 to 20. As shownherein, presence of an L group enables high TGase-mediated coupling whencycloalkynes are used.

Cycloalkynes, including specific compounds, are described for example inU.S. Pat. No. 7,807,619, the disclosure of which is incorporated hereinby reference.

In some embodiments, a cycloalkyne may be a compound of Formula A:

where:

R¹ is selected from a carbonyl, an alkyl ester, an aryl ester, asubstituted aryl ester, an aldehyde, an amide, an aryl amide, an alkylhalide, a thioester, a sulfonyl ester, an alkyl ketone, an aryl ketone,a substituted aryl ketone, and a halosulfonyl;

R¹ can be at any position on the cyclooctyne group other than at the twocarbons joined by the triple bond.

In some embodiments, the modified cycloalkyne is of Formula A, whereinone or more of the carbon atoms in the cyclooctyne ring, other than thetwo carbon atoms joined by a triple bond, is substituted with one ormore electron-withdrawing groups, e.g., a halo (bromo, chloro, fluoro,iodo), a nitro group, a cyano group, a sulfone group, or a sulfonic acidgroup. Thus, e.g., in some embodiments, a subject modified cycloalkyneis of Formula B:

where:

each of R² and R³ is independently: (a) H; (b) a halogen atom (e.g.,bromo, chloro, fluoro, iodo); (c)-W—(CH₂)_(n)—Z (where: n is an integerfrom 1-4 (e.g., n=1, 2, 3, or 4); W, if present, is O, N, or S; and Z isnitro, cyano, sulfonic acid, or a halogen); (d)-(CH₂)_(n)—W—(CH₂)_(m)—R⁴(where: n and m are each independently 1 or 2; W is O, N, S, orsulfonyl; if W is O, N, or S, then R⁴ is nitro, cyano, or halogen; andif W is sulfonyl, then R⁴ is H); or (e) —CH₂)_(n)—R⁴ (where: n is aninteger from 1-4 (e.g., n=1, 2, 3, or 4); and R⁴ is nitro, cyano,sulfonic acid, or a halogen); and

R¹ is selected from a carbonyl, an alkyl ester, an aryl ester, asubstituted aryl ester, an aldehyde, an amide, an aryl amide, an alkylhalide, a thioester, a sulfonyl ester, an alkyl ketone, an aryl ketone,a substituted aryl ketone and a halosulfonyl. R¹ can be at any positionon the cyclooctyne group other than at the two carbons linked by thetriple bond.

In one embodiment, R is a substituted or unsubstituted heterocyclicstrained alkyne. Cycloalkynes, including specific compounds, aredescribed for example in U.S. Pat. No. 8,133,515, the disclosure ofwhich is incorporated herein by reference. In one embodiment, the alkyneis of the Formula C:

wherein:

each R¹ is independently selected from the group consisting of hydrogen,halogen, hydroxy, alkoxy, nitrate, nitrite, sulfate, and a C₁-C₁₀ alkylor heteroalkyl;

each R² is independently selected from the group consisting of hydrogen,halogen, hydroxy, alkoxy, nitrate, nitrite, sulfate, and a C₁-C₁₀organic group; X represents CH—N—OR⁴, C—N—NR³R⁴, CHOR₄, or CHNHR₄; andeach R³ represents hydrogen or an organic group and R⁴ represents(C)_(n), or when present, X, L, V, Y or L′, V′, Y′ or Z of a linker ofthe invention.

Alkynes such as those described herein above can be reacted with atleast one 1,3-dipole-functional compound (e.g., embodied as an R′ moietyin a compound of Formula III) in a cyclization reaction to form aheterocyclic compound, preferably in the substantial absence of addedcatalyst (e.g., Cu(I)). A wide variety compounds having at least one1,3-dipole group attached thereto (having a three-atom pi-electronsystem containing 4 electrons delocalized over the three atoms) can beused to react with the alkynes disclosed herein. Exemplary 1,3-dipolegroups include, but are not limited to, azides, nitrile oxides,nitrones, azoxy groups, and acyl diazo groups.

The reactive moiety R is connected to L, or when present, V or Y, and isable to react with a suitable functional group (R′) on a reactionpartner, e.g. a complementary reagent of Formula III which undergoes ahigh conversion addition reaction when brought into contact with areactive moiety R. When reactive moiety R is present in an antibody ofFormula II, the reaction results in formation of an antibody of FormulaIV. In this reaction, the moieties R and R′ are transformed into themoiety (RR′). Any R′ moiety can be defined in the same way as a Rmoiety, so long as R and R′ are complementary when used in moieties thatare to be reacted together.

A compound of this invention may contain more than one reactive moietyR. The R moieties may or may not be the same. Any one of the R moietiesdisclosed herein can be utilized in Formula Ib and II. Any one of the Rmoieties described herein can be used in combination with any of the(C)_(n), X, L, V, Y, z, q, and r groups described herein. Any one of theR′ moieties disclosed herein can be utilized in Formula III. Any one ofthe R′ moieties described herein can be used in combination with any ofthe L′, V′, Y′, Z, z′, q′, and r′ groups described herein.

FIG. 1 shows reaction schemes for thio-maleimide additions, Staudingerligations, and Diels-Alder cycloadditions, where reactive groups oflinking reagents having a single reactive functionality combine withcomplementary reactive group attached to a therapeutic or diagnosticmoiety.

FIG. 2 shows reaction schemes for Diels-Alder cycloadditions and clickreactions where the reactive groups of linking reagents combine withcomplementary reactive group attached to an agent including atherapeutic, diagnostic, or other moiety.

It should be understood that, although not illustrated in FIGS. 1 and 2,the H₂NCH₂ group of the linking reagent may have undergone reaction withthe glutamine residue of a protein (e.g. antibody) prior to the highconversion addition reaction or that the aminomethylene may be in aprotected state. Alternatively, in other embodiments, the H₂NCH₂ groupof the linking reagent will not have undergone reaction with theglutamine residue of a protein (e.g. antibody) prior to the highconversion addition reaction or that the aminomethylene may be in aprotected state; in this case the linking reagent and reaction partnercan be used to conveniently form various combinations of linkers havingdifferent V, Y, and/or Z moieties that are ready to conjugate to anantibody.

The preparation of an exemplary linking reagent, according to anembodiment of the invention, and its conjugation with a protein isillustrated in FIG. 3, where: V and Y are absent, R is a thiol(sulfhydryl) reactive group that is ultimately generated from theS-acetyl protected thiol, SC(O)CH₃, r is 1; q is 1; z is 1; L is the twocarbon comprising framework C(O)CH₂; X is NH; (C)_(n) is (CH₂)₅; and Gis transformed from the (H₃C)₃COC(O) protecting group to H andultimately to the amide upon conjugation of a glutamine residue of aprotein. FIG. 4 illustrates the preparation of various exemplary linkingreagents, according to various embodiments of the invention, with asingle S-acetyl protected thiol reactive group that can be prepared froman N-succinimidyl-5-acetylthioester reagent. In addition to S-acetyl,other S-protecting groups can be employed, includingp-hydroxyphenylacyl, 2-quinoline, or Hqm and Hgm groups that can bedeprotected by the addition of hydrazine.

FIG. 5 illustrates the preparation of an exemplary linking reagent,according to an embodiment of the invention, and its conjugation with aprotein, where: V and Y are absent, R is an azide reactive group, r is1; q is 1; z is 1; L is the two carbon comprising framework C(O)CH₂; Xis NH; (C)_(n) is (CH₂)₅; and G is transformed from the (H₃C)₃COC(O)protecting group to H and ultimately to the amide upon conjugation of aglutamine residue of a protein. FIG. 6 illustrates the preparation ofvarious exemplary linking reagents, according to embodiments of theinvention, with a single azide reactive group that can be prepared froman N-succinimidyl-azide reagent.

FIG. 7 depicts the preparation of an exemplary linking reagent,according to an embodiment of the invention, and its conjugation with aprotein, where: V and Y are absent, R is an alkyne reactive group, r is1; q is 1; z is 1; L is a one carbon comprising framework CH₂; X is NH;(C)_(n) is (CH₂)₄—CH(CO₂H); and G is transformed from the (H₃C)₃COC(O)protecting group to H and ultimately to the amide upon conjugation of aglutamine residue of a protein. FIG. 8 shows the preparation of anexemplary linking reagent, according to an embodiment of the invention,and its conjugation with a protein, where: R is a norbornene reactivegroup, r is 1; q is 1; z is 1; L is the one carbon comprising frameworkC(O); X is NH; (C)_(n) is (CH₂)₄—CH(CO₂H); and G is transformed from the(H₃C)₃COC(O) protecting group to H and ultimately to the amide uponconjugation of a glutamine residue of a protein.

The selective and very high conversion addition reaction that can becarried out with the linking reagents, according to this aspect of theinvention, can be uncatalyzed or catalyzed reactions. For example, the2+4 Diels-Alder cycloadditions, thio-maleimide (or haloacetamide)additions, and Staudinger ligations can be carried out without acatalyst. Other very high conversion addition reactions, for example anyof the click reactions, can be catalyzed with metal salts, such as Cu,Ru, Ni, Pd, and Pt salts.

The linking group (RR′) in M of compounds of Formula IV represents theremainder of R when the reactive moiety R of Formula II has reacted witha reactive moiety R′ in a compound of Formula III. This group (RR′) thenlinks the moiety Z (e.g. comprised in the compound of formula IV) withL, V or Y. The group that remains may be a bond.

The V Moiety

The V moiety may be incorporated in the lysine-based linker (e.g.connected to L, optionally through Y). However, the V moiety may insteador in addition be incorporated in a compound comprising amoiety-of-interest Z (e.g. a compound R′—V—Y—Z of formula III) that willbe reacted with an antibody conjugated with a lysine-based linker toform an antibody conjugated to the moiety-of-interest Z. Any V′ moietycan be defined in the same way as a V moiety.

In the compounds of the invention, the V moiety is a group that iseither non-cleavable or conditionally cleavable, optionally after priorconditional transformation. In the latter case, it is designed to betransformed and/or cleaved from Y, or Z when Y is absent, by a chemical,photochemical, physical, biological, or enzymatic process, e.g. incertain conditions. This condition may for example comprise bringing acompound of the invention in an aqueous environment, which leads tohydrolysis of V, or bringing a compound of the invention in anenvironment that contains an enzyme that recognizes and cleaves V, orbringing a compound of the invention under reducing conditions, whichleads to reduction of V, or bringing a compound of the invention incontact with radiation, e.g., UV light, which leads to transformationand/or cleavage, or bringing a compound of the invention in contact withheat, which leads to transformation and/or cleavage, or bringing acompound of the invention under reduced pressure or bringing a compoundof the invention under elevated or high pressure, which leads totransformation and/or cleavage. This condition may further be met afteradministrating a compound of this invention to an animal, e.g., amammal: the condition may be met when the compound localizes to forexample a specific organ, tissue, cell, subcellular target, or microbialtarget, for example by the presence of internal factors (e.g.,target-specific enzymes or hypoxia) or application of external factors(e.g., radiation, magnetic fields) or the condition may already be metdirectly upon administration (e.g., enzymes). In general, transformationof V will directly or indirectly lead to cleavage of V from Y, or Z whenY is absent. It may occur that two or more separate transformationsand/or cleavages, requiring the same or different conditions, arerequired in order to cleave V completely from Y or Z. In this way,increased selectivity may be obtained. A compound of this invention maycontain more than one V moiety. These V moieties may or may not be thesame and may or may not require the same conditions for transformationand/or cleavage.

V may comprise for example a carbon comprising framework of 1 to 200atoms, optionally a carbon comprising framework of at least 10 atoms,e.g. 10 to 100 atoms or 20 to 100 atoms, substituted at one or moreatoms, optionally wherein the carbon comprising framework is a linearhydrocarbon or comprises a cyclic group, a symmetrically orasymmetrically branched hydrocarbon, monosaccharide, disaccharide,linear or branched oligosaccharide (asymmetrically branched orsymmetrically branched), other natural linear or branched oligomers(asymmetrically branched or symmetrically branched), an amino acid, adi-, tri-, tetra-, or oligopeptide, or more generally any dimer, trimer,or higher oligomer (linear, asymmetrically branched or symmetricallybranched) resulting from any chain-growth or step-growth polymerizationprocess.

Generally, V may be any straight, branched and/or cyclic C₂₋₃₀ alkyl,C₂₋₃₀ alkenyl, C₂₋₃₀ alkynyl, C₂₋₃₀ heteroalkyl, C₂₋₃₀ heteroalkenyl,C₂₋₃₀ heteroalkynyl, optionally wherein one or more homocyclic aromaticcompound radical or heterocyclic compound radical may be inserted;notably, any straight or branched C₂₋₅ alkyl, C₅₋₁₀ alkyl, C₁₁₋₂₀ alkyl,—O—C₁₋₅ alkyl, —O—C₅₋₁₀ alkyl, —O—C₁₁₋₂₀ alkyl, or (CH₂—CH₂—O—)₁₋₂₄ or(CH₂)_(x1)—(CH₂—O—CH₂)₁₋₂₄ —(CH₂)_(x2)-group, wherein x1 and x2 areindependently an integer selected among the range of 0 to 20, an aminoacid, an oligopeptide, glycan, sulfate, phosphate, or carboxylate.Optionally, V may be or absent. In some embodiments, V is a C₂₋₆ alkylgroup.

In one aspect of this invention, a compound of the invention is used totarget one or more therapeutic and/or diagnostic moieties Z to targetcells. In this instance, V may for example contain a substrate moleculethat is cleaved by an enzyme present in the vicinity of the target cellsor inside the target cells, for example tumor cells. V can for examplecontain a substrate that is cleaved by an enzyme present at elevatedlevels in the vicinity of or inside the target cells as compared toother parts of the body, or by an enzyme that is present only in thevicinity of or inside the target cells.

If target cell specificity is achieved solely based upon the selectivetransformation and/or cleavage of V at the target site, the condition(eventually) causing the cleavage should preferably, at least to acertain degree, be target cell-specific, whereas the presence of anothertarget-specific moiety in the compound of the invention, for instancewhen the antibody recognizes an antigen present on a target cell with adegree of specificity, reduces or takes away this requirement. Forexample, when an antibody causes specific internalization into a targetcell, an enzyme also present in other cells may transform and/or cleaveV. In one embodiment, transformation and/or cleavage of V occursintracellularly. In another embodiment, transformation and/or cleavageof V occurs extracellularly.

In one embodiment, the V moiety is a conditionally cleavable moiety.

In one embodiment, V contains a di-, tri-, tetra-, or oligopeptide whichconsists of an amino acid sequence recognized by a protease, for exampleplasmin, a cathepsin, cathepsin B, prostate-specific antigen (PSA),urokinase-type plasminogen activator (u-PA), or a member of the familyof matrix metalloproteinases, present in the vicinity of or inside thetarget cells, for example tumor cells. In one embodiment the inventionrelates to a conjugate wherein V is a dipeptide, tripeptide,tetrapeptide, or oligopeptide moiety comprised of natural L amino acids,unnatural D amino acids, or synthetic amino acids, or a peptidomimetic,or any combination thereof. In one embodiment, V is a peptide. Inanother embodiment, V is a dipeptide. In another embodiment, V is atripeptide. In another embodiment, V is a tetrapeptide. In yet anotherembodiment, V is a peptidomimetic.

In one embodiment, V contains a substrate for an enzyme.

In another embodiment, V contains a beta-glucuronide that is recognizedby beta-glucuronidase present in the vicinity of or inside tumor cells.

In one embodiment, V contains a substrate for an extracellular enzyme.In another embodiment, V contains a substrate for an intracellularenzyme.

In yet another embodiment, V contains a substrate for a lysosomalenzyme.

In yet another embodiment, V contains a substrate for the serineprotease plasmin.

In yet another embodiment, V contains a substrate for one or more of thecathepsins, for example cathepsin B. When V is cleaved extracellularly,the one or more Z moieties may be released extracellularly. This mayprovide the advantage that these Z moieties are not only able to affector detect the cell(s) directly surrounding the site of activation, butalso cells somewhat further away from the site of activation due todiffusion (bystander effect).

In one embodiment the invention relates to a compound wherein Vcomprises a tripeptide. The tripeptide may be linked via its C-terminusto Y. In one embodiment, the C-terminal amino acid residue of thetripeptide is selected from arginine, citrulline, and lysine, the middleamino acid residue of the tripeptide is selected from alanine, valine,leucine, isoleucine, methionine, phenylalanine, cyclohexylglycine,tryptophan and proline, and the N-terminal ammo acid residue of thetripeptide is selected from any natural or unnatural amino acid.

In another embodiment the invention relates to a compound wherein Vcomprises a dipeptide. The dipeptide may be linked via its C-terminus toY. In one embodiment, the C-terminal amino acid residue of the dipeptideis selected from alanine, arginine, citrulline, and lysine, and theN-terminal amino acid residue of the dipeptide is selected from anynatural or unnatural amino acid. In one embodiment, V is selected fromphenylalanine-lysine and valine-citrulline.

An example of a linker of the invention comprising a a lysine residue as(C)_(n) moiety and a valine-citrulline as the (V) moiety is shown below:

Optionally, the di-, tri-, tetra, or oligopeptide(s) comprise or consistor amino acids with non-negatively charged side chains (amino acidsother than aspartic acid or glutamic acid). Optionally, the di-, tri-,tetra, or oligopeptide(s) comprise or consist or amino acids selectedfrom amino acids with positively charged side chains, amino acids withpolar uncharged side chains, and amino acids with hydrophobic sidechains.

In another aspect of this invention, a compound of this invention isused to improve the pharmacokinetic properties of Z. V may in this casefor example be or contain a group that is cleaved by ubiquitous enzymes,e.g., esterases that are present in the circulation, by pH-controlledintramolecular cyclization, or by acid-catalyzed, base-catalyzed, ornon-catalyzed hydrolysis, or V may for example be or contain adisulfide. V may therefore, optionally together with the connecting atomof L and/or Y (or Z if Y is absent), for example form a carbonate,carbamate, urea, ester, amide, imine, hydrazone, oxime, disulfide,acetal, or ketal group. It is understood that V can also be or containsuch a moiety and/or be transformed and/or cleaved in the same or asimilar way when a compound of this invention is used for other purposesthan solely improving the pharmacokinetic properties of Z.

When the compounds of the invention are used for other purposes, e.g.,an ex vivo diagnostic assay, V may be or contain any of the moietiesmentioned above and transformation and/or cleavage of V may occur by anyone of the processes mentioned above or by any other functionaltransformation or cleavage process known to a person skilled in the art.For example, in a diagnostic assay, V may be cleaved or transformed byan enzyme, by reduction, or below, above, or at a certain pH.

When V is conditionally cleavable, the compounds of this invention aredesigned to eventually release at least one Z after cleavage andoptional prior transformation of V. Release of Z from a compound of thisinvention via another mechanism is however not excluded from thisinvention.

In any embodiment, V may contain a blocking group to prevent prematuretransformation and/or cleavage of V before the condition is met underwhich V is designed to be transformed and/or cleaved.

In another aspect of this invention, V is a moiety that isnon-cleavable. This means that V cannot be cleaved from Y, or Z when Yis absent, under the conditions the compound containing such a V moietyis designed to be applied, meaning that Z cannot be released in thisway. Release of Z from a compound of this invention via anothermechanism is however not excluded. When V is a non-cleavable moiety, Ymay optionally be absent. A non-cleavable V moiety may be any moietythat cannot be cleaved, or that can be cleaved only very slowly, underthe conditions the compound containing such a V moiety is designed to beapplied, e.g. in vivo or in vitro. For example, when applied in vivo, Vwill not or only very slowly be cleaved by enzymes present in the invivo model used or by hydrolysis or as a consequence of other biologicalprocesses that may occur in said model. Such V may therefore, optionallytogether with the connecting atom of L and/or Z, for example, be acarbonyl group, an amide group, an urea group, an ester group, acarbonate group, a carbamate group, or an optionally substitutedmethyleneoxy or methyleneamino group V may be preferred to benon-cleavable when it is not required that the one or more moieties Zare released. This may for example be the case when Z does not requireto become released before it can exert its therapeutic or diagnosticproperties.

In one embodiment V is connected to L via a functional group in the sidechain of one of the natural or unnatural amino acids. In anotherembodiment, the N-terminal amino acid of V is connected via its alphaamino group to L.

Any one of the V moieties disclosed herein can be utilized in FormulaIa, Ib, Ic, II, IVa and IVb. Any one of the V moieties described hereincan be used in combination with any of the (C)_(n), X, L, R, Y, Z, M, z,q, and r groups described herein. Any one of the V′ moieties disclosedherein can be utilized in Formula III. Any one of the V′ moietiesdescribed herein can be used in combination with any of the R′, V′, Y′,Z, z′, q′, and r′ groups described herein.

The Spacer System Y

The spacer system Y, when present, links V and optionally L to one ormore moieties R, and following reaction with a compound of Formula III,a moiety-of-interest Z. In one embodiment, Y is absent. In anotherembodiment, Y is a self-elimination spacer system. A spacer system Y maybe incorporated in a compound of this invention to for example improvethe properties of Z or the compound in general, to provide suitablecoupling chemistries, or to create space between V and Z. Any Y′ moietycan be defined in the same way as a Y moiety.

Spacer system Y may comprise for example a carbon comprising frameworkof 1 to 200 atoms, optionally a carbon comprising framework of at least10 atoms, e.g. 10 to 100 atoms or 20 to 100 atoms, substituted at one ormore atoms, optionally wherein the carbon comprising framework is alinear hydrocarbon or comprises a cyclic group, a symmetrically orasymmetrically branched hydrocarbon, monosaccharide, disaccharide,linear or branched oligosaccharide (asymmetrically branched orsymmetrically branched), other natural linear or branched oligomers(asymmetrically branched or symmetrically branched), an amino acid, adi-, tri-, tetra-, or oligopeptide, or more generally any dimer, trimer,or higher oligomer (linear, asymmetrically branched or symmetricallybranched) resulting from any chain-growth or step-growth polymerizationprocess.

Y may be any straight, branched and/or cyclic C₂₋₃₀ alkyl, C₂₋₃₀alkenyl, C₂₋₃₀ alkynyl, C₂₋₃₀ heteroalkyl, C₂₋₃₀ heteroalkenyl, C₂₋₃₀heteroalkynyl, optionally wherein one or more homocyclic aromaticcompound radical or heterocyclic compound radical may be inserted;notably, any straight or branched C₂₋₅ alkyl, C₅₋₁₀ alkyl, C₁₁₋₂₀ alkyl,—O—C₁₋₅ alkyl, —O—C₅₋₁₀ alkyl, —O—C₁₁₋₂₀ alkyl, or (CH₂—CH₂—O—)₁₋₂₄ or(CH₂)_(x1)—(CH₂—O—CH₂)₁₋₂₄ —(CH₂)_(x2)— group, wherein x1 and x2 areindependently an integer selected among the range of 0 to 20, an aminoacid, an oligopeptide, glycan, sulfate, phosphate, or carboxylate.Optionlly, Y is absent. In some embodiments, Y is a C₂₋₆ alkyl group.

A compound of this invention may contain more than one spacer system Y.These moieties Y may or may not be the same. In some embodiments thespacer system Y is a self-elimination spacer that is connected to one ormore other self-elimination spacers via a direct bond. Herein, a singleself-elimination spacer may also be referred to as a spacer system. Aspacer system may be branched or unbranched and contain one or moreattachment sites for Z as well as V. According to the invention,self-elimination spacers that are able to release only a single moietyare called ‘single release spacers’. Self-elimination spacers that areable to release two or more moieties are called ‘multiple releasespacers’. Spacers, may be either branched or unbranched andself-eliminating through a 1,2+2n-elimination (n>/=1), referred to as“electronic cascade spacers”. Spacers may eliminate through acyclization process under formation of a cyclic urea derivative,referred to as “ω-amino aminocarbonyl cyclization spacers”.

The spacer system Y may self-eliminating or non-self-eliminating. A“self-eliminating” spacer unit allows for release of the drug moietywithout a separate hydrolysis step. When a self-eliminating spacer isused, after cleavage or transformation of V, the side of Y linked to Vbecomes unblocked, which results in eventual release of one or moremoieties Z. The self-elimination spacer systems may for example be thosedescribed in WO 02/083180 and WO 2004/043493, which are incorporatedherein by reference in their entirety, as well as other self-eliminationspacers known to a person skilled in the art. In certain embodiments, aspacer unit of a linker comprises a p-aminobenzyl unit. In one suchembodiment, a p-aminobenzyl alcohol is attached to an amino acid unitvia an amide bond, and a carbamate, methylcarbamate, or carbonate ismade between the benzyl alcohol and a cytotoxic agent. In oneembodiment, the spacer unit is p-aminobenzyloxycarbonyl (PAB). Examplesof self-eliminating spacer units further include, but are not limitedto, aromatic compounds that are electronically similar to p-aminobenzylalcohol (see, e.g. US 2005/0256030 A1), such as2-aminoimidazol-5-methanol derivatives (Hay et al. (1999) Bioorg. Med.Chem. Lett. 9:2237) and ortho- or para-aminobenzylacetals. Spacers canbe used mat undergo cyclization upon amide bond hydrolysis, such assubstituted and unsubstituted 4-aminobutyric acid amides (Rodrigues etal. Chemistry Biology, 1995, 2, 223) and 2-aminophenylpropionic acidamides (Amsberry, et al., J. Org. Chem., 1990, 55. 5867). Elimination ofamine-containing drugs that are substituted at the a-position of glycine(Kingsbury, et al., J. Med. Chem., 1984, 27, 1447) are also examples ofself-immolative spacers.

A “non-self-eliminating” spacer unit is one in which part or all of thespacer unit remains bound to the moiety Z upon enzymatic (e.g.,proteolytic) cleavage of the antibody-moiety-of-interest conjugate.Examples of non-self-eliminating spacer units include, but are notlimited to, a glycine spacer unit and a glycine-glycine spacer unit.Other combinations of peptidic spacers susceptible to sequence-specificenzymatic cleavage are also contemplated. For example, enzymaticcleavage of an antibody-moiety-of-interest conjugate containing aglycine-glycine spacer unit by a tumor-cell associated protease wouldresult in release of a glycine-glycine-drug moiety from the remainder ofthe antibody-moiety-of-interest conjugate. In one such embodiment, theglycine-glycine-drug moiety is then subjected to a separate hydrolysisstep in the tumor cell, thus cleaving the glycine-glycine spacer unitfrom the drug moiety.

In a compound of this invention, a spacer system Y may be connected tomore than one V moiety. In this case, transformation and/or cleavage ofone of these V moieties may trigger the release of one or more Zmoieties. When V moieties that are transformed or cleaved underdifferent conditions are connected to the same Y, release of one or moreZ moieties may occur when a compound of this invention is brought underone of several different conditions.

Any one of the Y moieties disclosed herein can be utilized in FormulaIa, Ib, Ic, II, IVa and IVb. Any one of the Y moieties described hereincan be used in combination with any of the (C)_(n), X, L, V, Y, R, Z, M,z, q, and r groups described herein. Any one of the Y′ moietiesdisclosed herein can be utilized in Formula III. Any one of the Y′moieties described herein can be used in combination with any of the R′,L′, V′, Z, z′, q′, and r′ groups described herein.

Conjugation of Lysine-Based Linkers to an Antibody

TGases' transamidating activity was first observed in guinea-pig liver,and later in micro-organisms, plants, invertebrates, fish, amphibians,and mammals All TGs, except plant and bacterial TGs (referred to asBTG), require Ca2+ for activation. The Ca2+ concentrations required bymammalian TGases are normally in the supraphysiological range associatedwith most intracellular processes and Ca2+ activation is also modulatedby further regulatory processes, such that TGases are inactive undernormal conditions and only activated following major disruptions inphysiological homoeostatic mechanisms. Transglutaminases play animportant role in biological processes which are dependent on the rapidcovalent crosslinking of proteins, e.g. blood coagulation, skin-barrierformation and extracellular-matrix assembly. TGase-mediated reactionsresult in supramolecular protein structures with high rigidity andstability.

Enzymes of the TG-family catalyze covalent protein crosslinking byforming proteinase resistant isopeptide bonds between a lysine donorresidue of one protein and an acceptor glutamine residue of anotherprotein, and is accompanied by the release of ammonia. The catalyticmechanism of transglutaminases has been proposed as follows. After theGlycine-containing first substrate (acceptor or Q-substrate) binds tothe enzyme, it forms a γ-glutamylthioester with the cysteine residue inthe active center of TGase, known as the acylenzyme intermediate,accompanied by the release of ammonia. The second substrate (donor orK-substrate) then binds to the acylenzyme intermediate and attacks thethioester bond. The product (two proteins crosslinked by anNε(γ-glutamyl)lysine isopetide bridge) is formed and released. Thisre-establishes the active-centre Cys residue of the enzyme in itsoriginal form and allows it to participate in another cycle ofcatalysis. The formation of the covalent acylenzyme intermediate isthought to be the rate-limiting step in these reactions. The catalytictriad of many transglutaminases is papain-like, containing Cys-His-Asp(where His is histidine and Asp is aspartic acid) and, crucially, atryptophan (Trp) residue located 36 residues away from the active-centreCys. In contrast, bacterial TG isolated from Streptoverticillium sp(vide supra) has an atypical catalytic triad and shows no sequencehomology with the papain-like catalytic triad of other TGases.

TGases display strict specificity in recognition of glutamine proteinsubstrates. However, TGases display broad specificity for recognition ofthe acyl-acceptor amine group, which can either be the ε-amino group ofpeptidyl lysine or a low-molecular mass primary amine (frequently apolyamine) (see, e.g. Folk, et al. (1980) J. Biol. Chem. 255, 3695-3700.For example, in addition to lysine, the small lysine-mimicking primaryamine 5-pentylamine (cadaverin) and variants or fragments thereof canefficiently bind to the acylenzyme intermediate, and a pseudo-isopeptidebond with the glutamine-containing protein is formed. See, e.g., Lorand,L. et al. (1979) Biochemistry 18, 1756-1765 (1979); Murthy, S, N. et al.(1994). J. Biol. Chem. 269, 22907-22911 (1994); Murthy, P. et al. (2009)Biochemistry (2009).

Bacterial, archaeal and eukaryotic TGases have been characterized anddiffer in several ways from mammalian TGases (Lorand, L. & Graham, R. M.(2003) Nat. Rev. Mol. Cell. Biol. 4, 140-156). BTG and more generallymicrobial TGases (EC 2.3.2.13, protein-glutamine-γ-glutamyltransferase)such as Streptomyces mobaraensis are calcium-independent and have anamino acid sequence of) very different from those of mammalian TGs (Andoet al. (1989) Agric. Biol. Chem. 53, 2613-2617). BTG is furthermore muchsmaller (37.8 kDa versus 76.6 kDa for guinea pig liver TG).Additionally, BTG shows broader substrate specificity for the amineacceptor glutamine substrates in proteins than do mammalian TGases.These characteristics, together with a higher reaction rate, low cost ofproduction, and a decreased tendency to catalyze deamidation make BTG apreferred enzyme for use in industrial applications such as those of thepresent invention.

The antibodies that are to be conjugated to the lysine-based linker willpreferably be free of N-linked glycosylation (e.g. an antibody whichdoes not comprises glycosylation sites or a modified full-lengthantibody). Full-length wild-type IgG antibodies naturally compriseN-linked glycosylation at residue 297 of the heavy chain whichinterferes and prevents with TGase-mediated conjugation onto glutamineresidues in the CH2 domain. Consequently, antibodies may bedeglycosylated. Deglycosylation can be carried out as described hereinor according to any suitable method. For example, antibody (1 mg) in PBSbuffer (0.1 mol/L NaCl and 0.05 mol/L sodium phosphate buffer, pH 7.4)are incubated with 100 units (0.2 μL) of N-glycosidase F (PNGase F) fromFlavobacterium meningosepticum (New England BioLabs, Ipswich, UK) at 37°C. overnight. The enzyme is then removed by centrifugation-dialysis(Vivaspin MWCO 50 kDa, Vivascience, Winkel, Switzerland). The productcan be analyzed by LC/MS.

In one embodiment, the product is analyzed for drug loading (e.g. numberof conjugates per antibody. Such methods can be used to determine themean number of conjugates per antibody (e.g., the mean DAR) as well asthe distribution of number of conjugates per antibody in a composition,i.e. the percentage of total antibody with any given level of drugloading or DAR. The portion of antibodies having a number (n) ofconjugated acceptor glutamines (e.g. n=1, 2, 3, 4, 5, 6, etc.) can bedetermined. One technique adapted to such determination and moregenerally drug loading is hydrophobic interaction chromatography (HIC),HIC can be carried out as described for example in Hamblett et al.(2004) Cancer Res. 10: 7063-7070; Wakankar et al. (2011) mAbs 3(2):161-172; and Lyon et al (2012) Methods in Enzymology, Vol. 502: 123-138,the disclosure of which are incorporated herein by reference.

The method allows the application of any suitable type oftransglutaminase (TGase) for this purpose. Several types oftransglutaminases have been reported in various living organismsincluding microbials. Examples are TGase from guinea pig liver (GTGase),fish liver (FTGase) and microorganisms (MTGase) and any recombinantTGase (rTGase). Other TGases than the ones listed here can also be usedaccording to the invention. Examples of useful TGases include microbialtransglutaminases, such as e.g. from Streptomyces mobaraense,Streptomyces cinnamoneum and Streptomyces griseocarneum fall disclosedin U.S. Pat. No. 5,156,956, which is incorporated herein by reference),and Streptomyces lavendulae (disclosed in U.S. Pat. No. 5,252,469, whichis incorporated herein by reference) and Streptomyces ladakanum(JP2003199569, which is incorporated herein by reference). It should benoted that members of the former genus Streptoverticillium are nowincluded in the genus Streptomyces (Kaempfer, J Gen Microbiol, 137,1831-1892, 1991). Other useful microbial transglutaminases have beenisolated from Bacillus subtilis (disclosed in U.S. Pat. No. 5,731,183,which is incorporated herein by reference) and from various Myxomycetes.Other examples of useful microbial transglutaminases are those disclosedin WO 96/06931 (e.g. transglutaminase from Bacilus lydicus) and WO96/22366, both of which are incorporated herein by reference. Usefulnon-microbial transglutaminases include guinea-pig livertransglutaminase, and transglutaminases from various marine sources likethe flat fish Pagrus major (disclosed in EP-0555649, which isincorporated herein by reference), and the Japanese oyster Crassostreagigas (disclosed in U.S. Pat. No. 5,736,356, which is incorporatedherein by reference). A preferred TGase is bacterial transglutaminase(BTG) (see, e.g. EC 2.3.2.13, protein-glutamine-γ-glutamyltransferase).In a more preferred embodiment, the TGase is from S. mobaraense. Inanother embodiment, the TGase is a mutant TGase having at least 80%sequence homology with native TGase. A preferred example is recombinantbacterial transglutaminase derived from streptomyces mobaraensis(available from Zedira, Darmstadt, Germany)

The TGase-catalyzed reaction can be carried out under mild conditions,from several hours to a day (e.g. overnight). Recombinant BTG (EC2.3.2.13) from streptomyces mobaraensis (Zedira, Darmstadt, Germany) canbe used at a concentration of between 1 and 20 U/mL, preferably between6 U/mL and 20 U/mL. The lysine-based linker substrates are reacted withantibody (1 mg/mL) at ligand concentrations between 400 and 600 mol/L,providing a 60 to 90-fold excess of the substrates over the antibody, oroptionally at lower excess of substrates, e.g. 1- to 20-fold, or 10-20fold. The reactions are performed in potassium-free phosphate bufferedsaline (PBS; pH 8) at 37° C. After 4 h to several days (depending on theantibody and the ligand), steady-state conditions are achieved. Excessligand and enzyme are then removed using centrifugation-dialysis(Vivaspin MWCO 50 kDa, Vivascience, Winkel, Switzerland). Reactions aremonitored by LC/MS. Higher amounts of TGase can be used as a function ofdifferent lysine-derivatives and substrates.

An acceptor glutamine present on an antibody (e.g. part of theantibody's primary structure, including for example an antibody fragmentwith a peptide tag) will, under suitable conditions, be recognized by aTGase and covalently bound to a lysine-based linker (e.g., compound ofFormula I). The results is an antibody of Formula II (the acceptorglutamine is functionalized with the compound of Formula I). Resultingantibody conjugates can be analyzed using any suitable method.Preferably, the stoichiometry of the conjugated antibodies can becharacterized by liquid chromatography mass spectrometry (LC/MS) using atop-down approach in order to assess the number of lysine-based linkerand/or where applicable moieties-of-interest conjugated to antibodies,and in particular the homogeneity of the composition. Conjugates can bereduced before LC/MS analysis and light chains and heavy chains aremeasured separately.

Reaction Partners Comprising a Moiety-of-Interest Z and Reactive GroupR′

Once a lysine-based linker (e.g., compound of Formula I) comprising areactive moiety R is conjugated to an antibody (e.g., resulting in anantibody of Formula II) the antibody can be reacted with a compoundcomprising a moiety Z and a reactive group R′, thereby forming anantibody-moiety-of-interest conjugate. Typically, the conjugatedantibody (e.g. the antibody of Formula II) is subjected to adeprotection step to provide an unprotected reactive group (R) and theantibody is then reacted with a compound comprising a reaction partnerR′.

R′ is a reactive moiety, for example a moiety comprising an unprotectedor protected bioorthogonal-reaction compatible reactive group, forexample an unprotected or protected thiol, epoxide, maleimide,haloacetamide, o-phoshenearomatic ester, azide, fulminate, sulfonateester, alkyne, cyanide, amino-thiol, carbonyl, aldehyde, generally anygroup capable of oxime and hydrazine formation, 1,2,4,5-tetrazine,norbornene, other stained or otherwise electronically activated alkene,a substituted or unsubstituted cycloalkyne, generally any reactivegroups which form via bioorthogonal cycloaddition reaction a 1,3- or1,5-disubstituted triazole, any diene or strained alkene dienophile thatcan react via inverse electron demand Diels-Alder reaction, a protectedor unprotected amine, a carboxylic acid, an aldehyde, an oxyamine, solong as such group when unprotected is reactive with R (when R′ isunprotected).

When more than one R′ group is present in a compound of the formula, theR′ groups will preferably be compatible such that no R′ group is acomplementary reagent to any other R′ group. The L′, V′ and/or Y′ groupsof formulae I-IV can have r, q, and/or z sites of attachment for therespective V′, Y′, and R′ groups, where r and q represent the degree ofbranching or polymerization. The sites of attachment can comprise a bondor comprise a functional group selected from an alkene, alkyne, ether,thioether, ester, thioester, amine, amide, alkylamide, or otherfunctional group readily generated by a condensation or additionreaction.

In one embodiment, R′ is a moiety having a terminal alkyne or azide.

In one embodiment, R′ is a substituted or unsubstituted cycloalkyne, forexample a compound of Formula A:

where:

R¹ is selected from a carbonyl, an alkyl ester, an aryl ester, asubstituted aryl ester, an aldehyde, an amide, an aryl amide, an alkylhalide, a thioester, a sulfonyl ester, an alkyl ketone, an aryl ketone,a substituted aryl ketone and a halosulfonyl.

R¹ can be at any position on the cyclooctyne group other than at the twocarbons joined by the triple bond.

In some embodiments, the modified cycloalkyne is of Formula A, whereinone or more of the carbon atoms in the cyclooctyne ring, other than thetwo carbon atoms joined by a triple bond, is substituted with one ormore electron-withdrawing groups, e.g., a halo (bromo, chloro, fluoro,iodo), a nitro group, a cyano group, a sulfone group, or a sulfonic acidgroup. Thus, e.g., in some embodiments, a subject modified cycloalkyneis of Formula B:

where:

each of R² and R³ is independently: (a) H; (b) a halogen atom (e.g.,bromo, chloro, fluoro, iodo); (c)-W—(CH₂)_(n)—Z (where: n is an integerfrom 1-4 (e.g., n=1, 2, 3, or 4); W, if present, is O, N, or S; and Z isnitro, cyano, sulfonic acid, or a halogen); (d)-(CH₂)—, —W—(CH₂)_(m)—R⁴(where: n and m are each independently 1 or 2; W is O, N, S, orsulfonyl; if W is O, N, or S, then R⁴ is nitro, cyano, or halogen; andif W is sulfonyl, then R⁴ is H); or (e) —CH₂)_(n)—R⁴ (where: n is aninteger from 1-4 (e.g., n=1, 2, 3, or 4); and R⁴ is nitro, cyano,sulfonic acid, or a halogen); and

R¹ is selected from a carbonyl, an alkyl ester, an aryl ester, asubstituted aryl ester, an aldehyde, an amide, an aryl amide, an alkylhalide, a thioester, a sulfonyl ester, an alkyl ketone, an aryl ketone,a substituted aryl ketone and a halosulfonyl. R¹ can be at any positionon the cyclooctyne group other than at the two carbons linked by thetriple bond.

In one embodiment, R′ is a substituted or unsubstituted heterocyclicstrained alkyne. Cycloalkynes, including specific compounds, aredescribed for example in U.S. Pat. No. 8,133,515, the disclosure ofwhich is incorporated herein by reference. In one embodiment, the alkyneis of the Formula C:

wherein:

each R¹ is independently selected from the group consisting of hydrogen,halogen, hydroxy, alkoxy, nitrate, nitrite, sulfate, and a C₁-C₁₀organic group;

each R² is independently selected from the group consisting of hydrogen,halogen, hydroxy, alkoxy, nitrate, nitrite, sulfate, and a C₁-C₁₀organic group; X represents CH—N—OR₄, C—N—NR³R⁴, CHOR₄, or CHNHR₄; andeach R³ represents hydrogen or an organic group and R⁴ represents a bondthat attaches Formula C to Formula III.

Alkynes such as those described herein above can be reacted with atleast one 1,3-dipole-functional compound (e.g., embodied in reactivegroup R of Formula Ia or Ib in a cyclization reaction to form aheterocyclic compound, preferably in the substantial absence of addedcatalyst (e.g., Cu(I)).

In one embodiment, when R′ is a cycloalkyne, including a heterocycliccompound, the linking reagent of Formula Ia or Ib may comprise anon-cyclic R group, optionally furthermore wherein L is a bond or ashorter carbon framework as L group. For example, R may be a non-cyclicgroup and L may comprise a carbon framework of 1-5 linear carbon atoms,optionally substituted at one or more atoms.

Any one of the R′ moieties disclosed herein can be utilized in FormulaIII. Any one of the R′ moieties described herein can be used incombination with any of the L′, V′, Y′, Z, z′, q′, and r′ groupsdescribed herein.

The compounds of (e.g. Formula III) to be used in reaction with anantibody can be reacted with antibody (e.g., 1 mg/mL) at ligandconcentrations between 2 and 20 (or between 4 and 20) molar equivalentsto the antibody, optionally between 2 and 10 (or between 4 and 10) molarequivalents to the antibody, optionally at a less than, or about, 20,10, 5, 4 or 2 molar equivalents to the antibody. However it will beappreciated that higher excesses (equivalents of reaction partner (e.g.Formula III) to antibody (40 to 80 fold, 60 to 90-fold) can also beused.

The compounds of Formula III to be used in reaction with an antibodyconjugated to a lysine-based linker (but without a moiety-of-interest),e.g., an antibody of Formula II, as well as the resulting antibodyconjugates therefore comprise one or more moieties-of-interest Z. Thecompounds of Formula III may additionally comprise a moiety V and/or Y,typically depending on which elements are included in the lysine-basedlinker

The compounds of Formula III to be used in reaction with an antibodyconjugated to a lysine-based linker (e.g. an antibody of Formula II)will comprise moieties Z connected to linker L′ when Y′ and V′ areabsent, connected to the spacer system Y′ or, when Y′ is absent,connected to V′. Consequently, a compound of Formula III may comprise amoiety Z connected to or comprising a reactive group R′, optionally themoiety Z connected to a reactive group R′ via a spacer system Y′ or,when Y′ is absent, to a reactive group R′ via V′, or to a reactive groupR′ via a V′—Y′, wherein Z is preferably connected to Y′ and V′ isconnected to R′ and Y′.

A compound of Formula III may contain one, two or more Z moieties thatare the same or that differ from one another, e.g. different therapeuticmoieties, and/or diagnostic moieties.

In one embodiment, the antibody of Formula II is reacted with a compoundof Formula III comprising a moiety of interest Z comprising and areactive group R′ capable of forming a bond with reactive group R ofFormula Ib, Ic or II, optionally wherein the compound further comprisesa V′ and/or Y′ group. The compound comprising a moiety of interest Zcomprising and a reactive group R′ preferably comprises a structure ofFormula III, below,

R′-L′-(V′—(Y′—(Z)_(z′))_(q′))_(r′)  Formula III

where:

R′ is a reactive group, e.g. a reactive group complementary for formingat least one bond with reactive group R of Formula Ib, Ic or II;

L′ is a bond or a carbon comprising framework of 1 to 200 atomssubstituted at one or more atoms, optionally wherein the carboncomprising framework is a linear hydrocarbon, a symmetrically orasymmetrically branched hydrocarbon monosaccharide, disaccharide, linearor branched oligosaccharide (asymmetrically branched or symmetricallybranched), other natural linear or branched oligomers (asymmetricallybranched or symmetrically branched), or a dimer, timer, or higheroligomer (linear, asymmetrically branched or symmetrically branched)resulting from any chain-growth or step-growth polymerization process;

V′ is independently absent, a non-cleavable moiety or aconditionally-cleavable moiety that can optionally be cleaved ortransformed by a chemical, photochemical, physical, biological, orenzymatic process, cleavage of V ultimately leading to release of one ormore Z moieties. In some embodiments, V is, preferably, a di-, tri-,tetra-, or oligopeptide as described below in the section entitled “TheV Moiety”,

Y′ is independently absent or a spacer system (e.g., a self-eliminatingspacer system or a non-self-elimination spacer system) which iscomprised of 1 or more spacers,

Z is independently a reactive group (optionally protected) other than acomplementary reactive group for reaction with R′, a moiety thatimproves the pharmacokinetic properties, a therapeutic moiety, ordiagnostic moiety;

q′ and r′ are an integer selected among 1, 2, 3 or 4, representingdegree of branching; and

z′ is an integer selected among 1, 2, 3 or 4.

Where Z is a reactive group, it can be a moiety comprising anunprotected or protected thiol, maleimide, haloacetamide,o-phoshenearomatic ester, azide, fulminate, alkyne, cyanide, anthracene,1,2,4,5-tetrazine, norbornene, other stained or otherwise electronicallyactivated alkene or, optionally, a protected or unprotected amine when Xis absent and L, V, or Y is other than a bond or a continuation of abond. In an alternative embodiment Z can be a reactive moiety,preferably a moiety comprising an unprotected or protected thiol, anunprotected or protected amine, maleimide, haloacetamide,o-phoshenearomatic ester, azide, fulminate, alkyne, cyanide, anthracene,1,2,4,5-tetrazine, norbornene, other stained or otherwise electronicallyactivated alkene. Preferably R is not an amine when n=5 and X, L, V andY are absent. Preferably R is not an amine when n=4 and X, L, V and Yare absent.

The moiety R′ is connected to Z, or optionally to Z via V′ and/or Y′ andis able to react with a suitable functional group R on a reactionpartner, e.g. group R on the lysine-based linker of formula Ib, Ic orII. As discussed above, when the reactive moiety R′ is designed to reactwith a reactive group R, a compound of Formula Ic or IVb is formed.

The L′ group can be a carbon comprising framework, where L is asymmetrically or asymmetrically branched hydrocarbon, monosaccharide,disaccharide, oligosaccharide, other natural oligomer, dimer, trimer, orhigher oligomer resulting from any chain-growth or step-growthpolymerization process, wherein L′ has r′, q′, and/or z′ sites ofattachment for the respective V′, Y′, and R′ groups, where r′ and q′represent the degree of branching or polymerization. The sites ofattachment can comprise a bond or comprise a functional group selectedfrom an alkene, alkyne, ether, thioether, ester, thioester, amine,amide, alkylamide, or other functional group readily generated by acondensation or addition reaction.

The linking group (RR′) in M of compounds of Formula (Ic) and (IVb)represents the R′ addition product of a reactive moiety R′ and areactive moiety R. This group then links the moiety Z) with L, V or Y,preferably via (RR′) of M is L′, V′, and/or Y′. The group that remainsmay be a bond. Typically, however, L′, V′, and/or Y′ is a linking group.RR′ can be an addition product of a: thio-maleimide (or haloacetamide)addition, for example, a N,S-disubstituted-3-thio-pyrrolidine-2,5-dione;Staudinger ligation, for example, a N,3- orN,4-substitued-5-dipenylphosphinoxide-benzoic amide; Huisgen1,3-cycloaddition (click reaction), for example, aN,S-disubstituted-3-thio-pyrrolidine-2,5-dione,1,4-disubstituted-1,2,3-triazole, 3,5-disubstituted-isooxazole, or3,5-disubstituted-tetrazole; Diels-Alder cycloaddition adduct, forexample the 2,4-cycloaddition product between an O orN-substituted-5-norbornene-2-carboxylic ester or amide,N-substituted-5-norbornene-2,3-dicarboxylic imide, O orN-substituted-7-oxonorbornene-5-carboxylic ester or amide, orN-substituted-7-oxonorbornene-5,6-dicarboxylic imide and a 9-substitutedanthracene or 3-substituted 1,2,4,5-tetrazine; or any high yieldselective amidation or imidization reaction. Some reactions and the RR′reaction products are illustrated in FIGS. 1 and 2.

Examples of compounds of Formula III include but are not limited tocompound having the R′, L′, V′, Y′ and Z groups shows in Table 4 herein.Examples of compounds of Formula III include but are not limited tocompound having the R′, L′, V′, Y′ and Z groups shows in Table 3 herein.The symbol (−) in the tables indicates that the particular R′, L′, V′,Y′ or Z is absent. V and Y groups, for example, can comprise anystructural features in the sections titled “The V Moiety” and “The YMoiety” herein. The L, V and/or Y groups of Formual III represented inTable 4 can have r′, q′, and/or z′ sites of attachment for therespective V, Y, and R or Z groups, where r and q represent the degreeof branching or polymerization; r′, q′, and/or z′ can be selected from1, 2, 3 or 4.

Non-limiting examples of compounds of Formula Ia and reaction partnersof Formula III are shown in Table 5.

The invention thus includes, in one embodiment, a method comprisingreacting a compound of Formula II with a compound for Formula III toobtain a compound of Formula IVb.

It will be appreciated that different configurations of reactionspartners (i.e. antibodies having a functionalized glutamine of FormulaII and compound for Formula III) can be envisaged. For example, in somecases it may be advantageous to maintain a particular linker and spacersystem and evaluate different moieties Z for their effect on anantibody, in which case the lysine-based linker may comprise L-V—Y—R andthe reaction partner will not comprise V—Y (e.g. the reaction partnerwill comprise R′-Z. An example of configurations is shown in Table 1.The invention also provides exemplary methods of evaluating a V, Yand/or Z moiety comprising: (a) reacting an antibody having afunctionalized glutamine of Formula II of rows 1-10 with two, three,four or more compounds of Formula III of the respective row 1-10,wherein the compounds of Formula III differ in their V, Y and/or Zmoiety, and (b) evaluating the effect of said differing V, Y and/or Zmoiety on the antibodies (e.g. yield and stoichiometry of coupling,biological activity, stability or generally any pharmacologicalproperties). For each respective row 1-10, the method may becharacterized as a method for evaluation according to column 4“Exemplary evaluation methods”.

TABLE 1 Functionalized acceptor Reaction glutamine of antibody partnerExemplary evaluation (Formula II) (Formula III) method 1(Q)-NH-(C)_(n)-X-L-(V- R′-L′-(Z)_(z′) Evaluating Z moieties in((Y)-(R)_(z))_(q))_(r) the context of a particular V-Y 2(Q)-NH-(C)_(n)-X-L-(V- R′-L′-(Y′-(Z)_(z′))_(q′) Evaluating Y′ moietiesor (R)_(z))_(r) Y′-Z couples in the context of a particular V 4(Q)-NH-(C)_(n)-X-L-(V- R′-L′-(Z)_(z′) Evaluating Z moieties in(R)_(z))_(r) the context of a particular V 5 (Q)-NH-(C)_(n)-X-L-(Y-R′-L′-(Z)_(z′) Evaluating Z moieties in (R)_(z))_(r) the context of aparticular Y 6 (Q)-NH-(C)_(n)-X-L-(R)_(z) R′-L′-(Y′-(Z)_(z′))_(q′)Evaluating Y′ moieties or Y′-Z couples 7 (Q)-NH-(C)_(n)-X-L-(R)_(z)R′-L′-(V′-(Z)_(z′))_(r′) Evaluating V′ moieties or V′-Z couples 8(Q)-NH-(C)_(n)-X-L-(R)_(z) R′-L′-(Z)_(z′) Evaluating Z moieties 9(Q)-NH-(C)_(n)-X-L-(R)_(z) R′-(Z)_(z′) Evaluating Z moieties 10(Q)-NH-(C)_(n)-X-L-(R)_(z) R′-L′-(V′-(Y′- Evaluating Y′ moieties, V′(Z)_(z′))_(q′))_(r)′ moieties, or V′-Y′-Z couples

The step of reacting an antibody having a lysine-based linker (e.g.,compound of Formula Ib or Ic) comprising a reactive moiety R conjugatedthereto with a compound comprising a moiety Z and a reactive group R′ toform an antibody-moiety-of-interest conjugate can advantageously becarried out by binding the antibody onto a solid support. Use of a solidsupport for this step can allow for antibody samples of differentinitial concentrations and amounts to be reacted and then compared foractivity. Use of a solid support also permits improved purification offunctionalized antibodies. Finally, use of a solid support for this stepallows an increase in efficiency in production and/or increase incompletion of reactions because the compound comprising a moiety Z and areactive group R′ can be recovered and then reintroduced to the solidsupport; this may reduce loss of expensive reagents such as cytotoxicdrugs.

The amount of antibody used in solid-support based methods may be smallamounts (e.g., 1 to 500 μg) of antibody.

Generally, the solid support may be any suitable insoluble,functionalized material to which the antibodies can be reversiblyattached, either directly or indirectly, allowing them to be separatedfrom unwanted materials, for example, excess reagents, contaminants, andsolvents. Examples of solid supports include, for example,functionalized polymeric materials, e.g., agarose, or its bead formSepharose®, dextran, polystyrene and polypropylene, or mixtures thereof;compact discs comprising microfluidic channel structures; protein arraychips; pipet tips; membranes, e.g., nitrocellulose or PVDF membranes;and microparticles, e.g., paramagnetic or non-paramagnetic beads. Insome embodiments, an affinity medium will be bound to the solid supportand the antibody will be indirectly attached to solid support via theaffinity medium. In one aspect, the solid support comprises a protein Aaffinity medium or protein G affinity medium. A “protein A affinitymedium” and a “protein G affinity medium” each refer to a solid phaseonto which is bound a natural or synthetic protein comprising anFc-binding domain of protein A or protein G, respectively, or a mutatedvariant or fragment of an Fc-binding domain of protein A or protein G,respectively, which variant or fragment retains the affinity for anFc-portion of an antibody.

The present methods can comprise a step of immobilizing an antibodycomprising a lysine-based linker (e.g., compound of Formula Ia or Ib)comprising a reactive moiety R conjugated thereto on a solid support toprovide an immobilized antibody. In some embodiments, the solid supportwill have the capacity to bind more antibody than the amount present inthe antibody-containing sample or, in other words, the amount ofantibody bound to the solid support following the immobilization stepwill be less than the capacity of the solid support. Because the samplesgenerally vary with respect to antibody quantity, there will becorresponding variability in the amount of immobilized antibody from onesample as compared to another.

It will be possible to optionally limit the quantity of bound antibodyand the solid support will only have the capacity to bind up to acertain amount of antibody (e.g., up to 5 μg, up to 10 μg, or up to 15μg of protein). In these embodiments, although there will be a limit asto the maximum amount of antibody that can be bound to the solidsupport, there may still be variability in the amount of immobilizedantibody in one sample as compared to another. This is because one ormore of the samples might contain a small quantity of antibody, lessthan the maximum loading capacity of the solid support. One approach forpreparing a solid support that has limited capacity for binding antibodyis to make a very low-capacity resin such that a larger volume of resinslurry (20 uL for example) contains only enough capacity to bind 5 ug ofantibody. An alternative approach is to reduce the effective capacity ofa resin by diluting the resin with an appropriate volume ofnon-functionalized resin. For example, a protein G-sepharose resin witha binding capacity of 20 ug/uL could be converted to a mixed resin withan effective binding capacity of 0.5 ug/uL by mixing 1 part of proteinG-sepharose with 40 parts unfunctionalized sepharose. In performing sucha resin dilution, in some embodiments, the diluent will be a resin whichis constructed from the same base material as the affinity resin, haspore sizes small enough to exclude antibodies, and lacks any surfacefunctionality which may interact with antibodies or the chemicalreagents used to prepare antibody conjugates.

Antibodies are generally immobilized on a solid support by the step ofapplying an antibody-containing sample to a solid support. If desired, awashing step can be performed following immobilization to separate theimmobilized antibodies from the cell culture supernatant or othercomponents of the antibody-containing samples.

Once the antibodies are immobilized on the solid support, the conjugatedantibody (e.g. the antibody of Formula II) is typically subjected to adeprotection step to provide an unprotected reactive group (R) and theantibody is then reacted with a compound comprising a reaction partnerR′. A reaction step is then performed comprising applying a compoundcomprising a moiety Z and a reactive group R′ (e.g. a compound ofFormula III) to a solid support to generate anantibody-moiety-of-interest conjugate (e.g., antibody of Formula IVb).

In some embodiments of the present invention, the compound comprising amoiety Z and a reactive group R′ will be provided in molar excess (molarexcess as to the reactive groups (R)).

After contacting the reduced antibodies with the appropriate amountcompound comprising reactive group (R′), a washing step can be performedto remove any unreacted materials. Optionally, unreacted compoundcomprising a moiety Z and a reactive group R′ is recovered; optionally,unreacted compound is re-applied to the solid support to provide forhigher completion of the reaction between antibody comprising reactivegroup (R) and compound comprising reactive group (R′).

Subsequently, the immobilized antibody conjugates can be eluted from thesolid support to provide antibody conjugate compositions. Methods ofeluting proteins from solid supports are known in the art and theskilled practitioner will be able to select an appropriate buffer forelution. For example, in embodiments, where the solid support comprisesprotein A or protein G resin, the antibody conjugates can be eluted withstandard low pH buffers for elution from protein A or protein G columns

The Moiety Z

The moieties Z can be connected to Y or Y′ or, when absent, to V or V′,or, when absent, to L or, when absent to X, or when absent to (C)_(n).Connections to Y, V or L may optionally be via R or RR′. Connection maybe via any suitable atoms. In one embodiment, Z is coupled via oxygen(from for example a hydroxyl group or carboxyl group), carbon (from forexample a carbonyl group), nitrogen (from for example a primary orsecondary amino group), or sulfur (from for example a sulfhydryl group).In one embodiment, Z is coupled in the compounds of this invention via agroup such that its therapeutic abilities or diagnostic characteristicsare, at least partly, blocked or masked. In case a compound of theinvention is to be used for treating or preventing disease in an animal,e.g., a mammal, the Z moieties are generally therapeutic moieties. Incase a compound of the invention is used to make a diagnosis or used inan ex vivo or in vivo diagnostic assay, the Z moieties are generallydiagnostic moieties, for example chromogenic, fluorogenic,phosphorogenic, chemiluminescent, or bio luminescent compounds.

In one embodiment, the Z moiety is compound, preferably an organiccompound, having a molecular weight of at least 300 g/mol, 400 g/mol,500 g/mol, 600 g/mol, 700 g/mol, 800 g/mol, 900 g/mol, 1000 g/mol or2000 g/mol.

In one embodiment, the Z moiety is a chemical compound displayinghydrophobic properties, optionally additionally having a molecularweight of at least 300 g/mol, 400 g/mol, 500 g/mol, 600 g/mol, 700g/mol, 800 g/mol, 900 g/mol. 1000 g/mol or 2000 g/mol. Hydrophobiccharacter may be determined, for example, by decreased water solubility,decreased polarity, decreased potential for hydrogen bonding, and/or anincreased oil/water partition coefficient. The presently disclosedmethods can be used to produce antibody conjugates where moiety ofinterest (Z) comprises a hydrophobic drug. As used herein, the term“hydrophobic” is a physical property of a molecule that is repelled froma mass of water. Hydrophobic compounds can be solubilized in nonpolarsolvents, including but not limited to, organic solvents. Hydrophobicitycan be conferred by the inclusion of apolar or nonpolar chemical groupsthat include, but are not limited to, saturated and unsaturatedaliphatic hydrocarbon groups and such groups substituted by one or morearomatic, cycloaliphatic or heterocyclic group(s). Conversely,“hydrophilic” molecules are capable of hydrogen bonding with a watermolecule and are therefore soluble in water and other polar solvents.The terms “hydrophilic” and “polar” can be used interchangeably.Hydrophilic characteristics derive from the presence of polar or chargedgroups, such as carbohydrates, phosphate, carboxylic, sulfato, amino,sulfhydryl, nitro, hydroxy and other like groups.

Hydrophobic molecules are poorly water soluble, for example, having asolubility of less than about 10 mg/ml. In some embodiments, thehydrophobic compound can have a solubility of less than about 1 mg/ml inwater. In other embodiments, the hydrophobic compound has solubility inwater of less than about 50, ng/ml, 10 ng/ml, and in particularembodiments, about 1 ng/ml or 2.5 ng/ml. In other embodiments, thehydrophobic compound can have a solubility of about 0.001 ng/ml to about10 mg/ml, including but not limited to 0.001 ng/ml, 0.01 ng/ml, 0.1ng/ml, 1 ng/ml, 2 ng/ml, 5 ng/ml, 10 ng/ml, 50 ng/ml, 100 ng/ml, 500ng/ml, 1 mg/ml, 5 mg/ml, and 10 mg/ml, and any other concentrationbetween 0.001 ng/ml and 10 mg/ml.

Representative, non-limiting examples of hydrophobic drugs that can beformulated using the presently disclosed methods include taxanes, e.g.paclitaxel (PTX), and camptothecin (CPT), maytansanoids, duocarmycins,dolastatins and auristatins. Such drugs are poorly soluble in water,e.g. PTX has a solubility in water of less than about 1 ng/ml, CPT has awater solubility of about 2.5 ng/ml. Linkers and modified antibodies ofthe invention can advantageously link hydrophobic drugs to antibodies.

In other embodiments, in view of hydrophobic drugs being poor substratesfor TGase (in the absence of improved linkers or modified antibodies ofthe invention), the Z mioety may advantageously be a hydrophilic drug.Examples of hydrophilic drugs include amatoxins. Amatoxins are cyclicpeptides composed of 8 amino acids as isolated from the genus Amanita.Amatoxins also include a range of chemical derivatives, semisyntheticanalogs and synthetic analogs built from building blocks according tothe master structure of the −5 natural compounds (cyclic, 8 aminoacids),synthetic or semisynthetic analogs containing non-hydroxylated aminoacids instead of the hydroxylated amino acids, synthetic orsemisynthetic analogs, in which the thioether sulfoxide moiety isreplaced by a sulfide, sulfone, or by atoms different from sulfur, e.g.a carbon atom as in a carbaanalog of amanitin. Functionally, amatoxinsare defined as peptides or depsipeptides that inhibit mammalian RNApolymerase II. Preferred amatoxins are those with a functional group(e.g. a carboxylic group, an amino group, a hydroxy group, a thiol or athiol-capturing group) that can be reacted with linker molecules orproteins. Amatoxins are described for example in European Patentpublication no. 1859811, PCT publication nos. WO2010/115630 andWO2012/041504).

In one embodiment, the Z moiety is a large compound (e.g., molecularweight of at least 300 g/mol, 400 g/mol, 500 g/mol, 600 g/mol or 700g/mol) comprising a polycyclic group, tricycle or one or moremacrocycles. Such groups are often typical of hydrophobic and/or rigidstructures. Examples of cytotoxic drugs that comprise a macrocycle (e.g.a ring of nine or more atoms) include maytansinoids, amatoxins,epothilones and taxanes. In one embodiment, the Z moiety comprises aring of 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 atoms, or between 9 and200 atoms. In one embodiment, the Z moiety is a chemical compound havinga negative charge, optionally additionally displaying hydrophobicproperties and/or having a molecular weight of at least 300 g/mol, 400g/mol, 500 g/mol, 600 g/mol, 700 g/mol, 800 g/mol, 900 g/mol, 1000 g/molor 2000 g/mol.

When more than one Z moiety is connected to a self-elimination spacersystem Y or Y′, at least one Z should be released upon self-eliminationof Y or Y′. The moiety Z initially released may be a moiety that is nota fully active moiety itself. In other words, Z may be a moiety that haslimited diagnostic or therapeutic abilities, e.g. a moiety that acts asa prodrug. Such a Z moiety may require further processing or metabolism,e.g., hydrolysis, enzymatic cleavage, or enzymatic modification (forexample phosphorylation, reduction, or oxidation) in order to becomefully active. In one embodiment, such further processing isintentionally designed for Z to for example allow Z to reach its finaltarget or cross a biological barrier, e.g., a cell membrane or a nuclearmembrane, before it is fully activated. Z may for example contain ahydrophobic moiety that enables Z to cross a cell membrane. Thishydrophobic moiety may then be hydrolyzed or removed in any other wayintracellularly.

In one aspect of the invention, a Z moiety may be a backbone (e.g.polymer) to which a plurality of drugs or diagnostic moieties arelinked. For example, Z may be a polyacetal- or polyacetalderivative-based polymer comprising a plurality of drug molecules, see,e.g., Yurkovetskiy et al. (2004) Mol. Pharm. 1(5): 375-382 and WO2011/120053, the disclosures of which are incorporated herein byreference; for example Z may be a polymer compound of Formula I of WO2011/120053 comprising a plurality of cytotoxic anti-cancer agents.

In one aspect of this invention, one or more moieties Z are eachselected from a therapeutic or diagnostic agent.

In another embodiment of this invention, one or more moieties Z are eacha therapeutic agent.

In another embodiment of this invention, all moieties Z are each atherapeutic agent.

In yet another embodiment, the moieties Z each are the same therapeuticmoiety.

In yet another embodiment, the moieties Z comprise at least twodifferent therapeutic moieties.

The moiety Z includes, for example, antineoplastic agents, drugs, toxins(such as enzymatically active toxins of bacterial or plant origin andfragments thereof e.g. ricin and fragments thereof) biologically activeproteins, for example enzymes, other antibody or antibody fragments,synthetic or naturally occurring polymers, nucleic acids and fragmentsthereof e.g. DNA, RNA and fragments thereof, radionuclides, particularlyradioiodide, radioisotopes, chelated metals, nanoparticles and reportergroups such as fluorescent compounds or compounds which may be detectedby NMR or ESR spectroscopy.

In one embodiment, the one or more moieties Z are each independentlychosen from an antibiotic, an anti-bacterial agent, an antimicrobialagent, an anti-inflammatory agent, an anti-infectious disease agent, ananti-autoimmune disease agent, an anti-viral agent, or an anticanceragent, preferably a cytotoxic anti-cancer agent.

In another embodiment, the one or more moieties Z are each an anticanceragent. In a further embodiment, the one or more moieties Z are each ahydroxyl-containing anticancer agent.

In one embodiment, Z is an alkylating agent, preferably a DNA alkylatingagent. An alkylation agent is a compound that can replace a hydrogenatom with an alkyl group under physiological conditions (e.g. pH 7.4, 37C, aqueous solution). Alkylation reactions are typically described interms of substitution reactions by N, O and S heteroatomic nucleophileswith the electrophilic alkylating agent, although Michael additionreactions are also important. Examples of alkylating agents includenitrogen and sulfur mustards, ethylenimines, methanosulfonates, CC-1065and duocarmycins, nitrosoureas, platinum-containing agents, agents thateffectuate Topoisomerase II-mediated site dependent alkylation of DNA(e.g. psorospermin and related bisfuranoxanthones), ecteinascidin andother or related DNA minor groove alkylation agents.

In one embodiment, Z is a DNA minor groove binding and/or alkylatingagent, e.g, a pyrrolobenzodiazepine, a duocarmycin, or derivativesthereof.

In a further embodiment, the one or more moieties Z are eachindependently selected from the group consisting of taxanes,anthracyclines, camptothecins, epothilones, mytomycins, combretastatins,vinca alkaloids, nitrogen mustards, maytansinoids, calicheamycins,duocarmycins, tubulysins, dolastatins and auristatins, enediynes,amatoxins, pyrrolobenzodiazepines, ethylenimines, radioisotopes,therapeutic proteins and peptides, and toxins or fragments thereof.

In a further embodiment, the one or more moieties Z are eachindependently selected from cyclophosphamide, ifosfamide, chlorambucil,4-(bis(2-chloroethyl)amino)phenol, 4-(bis(2-fluoroethyl)ammo)phenol,N,N-bis(2-chloroethyl)-p-phenylenediamine,N,N-bis(2-fluoro-ethyl)-p-phenylenediamine, carmustine, lomustine,treosulfan, dacarbazine, cisplatin, carboplatin, vincristine,vinblastine, vindesine, vinorelbine, paclitaxel, docetaxel, etoposide,teniposide, topotecan, inirotecan, 9-aminocamptothecin,9-nitrocamptothecin, 10-hydroxycamptothecin, lurtotecan, camptothecin,crisnatol, mitomycin C, mitomycin A, methotrexate, trimetrexate,mycophenolic acid, tiazofurin, ribavirin, hydroxyurea, deferoxamine,5-fluorouracil, floxuridine, doxifluridine, raltitrexed, cytarabine,cytosine arabinoside, fludarabine, 6-mercaptopurine, thioguanine,raloxifen, megestrol, goserelin, leuprolide acetate, flutamide,bicalutamide, vertoporfin, phthalocyanine, photosensitizer Pc4,demethoxy-hypocrellin A, interferon-alpha, interferon-gamma, tumornecrosis factor, lovastatin, staurosporine, actinomycin D, bleomycin A2,bleomycin B2, peplomycin, daunorubicin, doxorubicin,N-(5,5-diacetoxypentyl)doxorubicin, morpholino doxorubicin, idarubicin,epirubicin, pirarubicin, zorubicin, mitoxantrone, thapsigargin,N⁸-acetylspermidine, tallysomycin, esperamycin, butyric acid, retinoicacid, 1,8-dihydroxybicyclo[7.3.1]trideca-4-ene-2,6-diyne-13-one,anguidine, podophyllotoxin, combretastatin A-4, pancratistatin,tubulysin A, tubulysin D, caminomycin, streptonigrin, elliptmiumacetate, maytansine, maytansinol, calicheamycin, mertansine (DM1),N-acetyl-γ₁ ¹-calicheamycin, calicheamycin-γ₁ ^(I), calicheamycin-α₂ ¹,calicheamycin-α₃ ¹, duocarmycin SA, duocarmycin A, CC-1065, CBI-TMI,duocarmycin C2, duocarmycin B2, centanamycin, dolastatin, auristatin E,monomethylauristatin E (MMAE), monomethylauristatin F (MMAF),α-amanitin, α-amanitin, γ-amanitin, ε-amanitin, amanin, amaninamide,amanullin, and amanullinic acid and derivatives thereof.

Exemplary auristatin embodiments include the N-terminus linkedmonomethylauristatin drug moieties comprising a structure of any ofFormulas V and VI below:

wherein the wavy line of V and VI indicates the covalent attachment siteto a L, L′, V, V′, Y, Y′, (RR′), R′ or (C)_(n) group of a compound ofthe invention (e.g. a compound of Formula I, II or IV), andindependently at each location:

R² is selected from H and C₁-C₈ alkyl;

R³ is selected from H, C₁-C₈ alkyl, C₁-C₈ carbocycle, aryl, C₁-C₈alkyl-aryl, C₁-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈ heterocycle and C₁-C₈alkyl-(C₃-C₈ heterocycle);

R⁴ is selected from H, C₁-C₈ alkyl, C₃-C₈ carbocycle. aryl, C₁-C₈alkyl-aryl, C₁-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈ heterocycle and C₁-C₈alkyl-(C₃-C₈ heterocycle);

R⁵ is selected from H and methyl;

or R⁴ and R⁵ jointly form a carbocyclic ring and have the formula—(CR^(a)R^(b))_(n) wherein R^(a) and R^(b) are independently selectedfrom H, C₁-C₈ alkyl and C₃-C₈-carbocycle and n is selected from 2, 3, 4,5 and 6;

R⁶ is selected from H and C₁-C₈ alkyl;

r⁷ is selected from H, C₁-C₈ alkyl, C₃-C₈ carbocycle, aryl, C₁-C₈alkyl-aryl, C₁-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈ heterocycle and C₁-C₈alkyl-(C₃-C₈ heterocycle);

each R⁸ is independently selected from H, OH, C₁-C₈ alkyl, C₃-C₈carbocycle and O—(C₁-C₈ alkyl);

R⁹ is selected from H and C₁-C₈ alkyl;

R¹⁹ is selected from aryl or C₃-C₈ heterocycle;

Z is O, S, NH, or NR¹² wherein R¹² is C₁-C₈ alkyl;

R¹¹ is selected from H, C₁-C₂₀ alkyl, aryl, C₃-C₈ heterocycle,—(R¹³O)_(m)—R¹⁴, or —(R¹³O)_(m)—CH(R¹⁵)₂; m is an integer ranging from1-1000;

R¹³ is C₂-C₈ alkyl;

R¹⁴ is H or C₁-C₈ alkyl;

each occurrence of R¹⁵ is independently H, COOH, —(CH₂)_(n)—N(R¹⁶)₂,—(CH₂)_(n)—SO₃—C₁-C₈ alkyl;

each occurrence of R¹⁶ is independently H, C₁-C₈ alkyl, or—(CH₂)_(n)—COOH;

R¹⁸ is selected from —C(R⁸)₂—C(R⁸)₂-aryl, —C(R⁸)₂—C(R⁸)₂—C₃-C₈heterocycle), and —C(R⁸)₂—C(R⁸)₂—(C₃-C₈ carbocycle); and

n is an integer ranging from 0 to 6.

In one embodiment, R³, R⁴ and R⁷ are independently isopropyl orsec-butyl and R⁵ is —H or methyl. In an exemplary embodiment. R³ and R⁴are each isopropyl, R⁵ is —H, and R⁷ is sec-butyl.

In yet another embodiment, R² and R⁶ are each methyl, and R⁹ is —H.

In still another embodiment, each occurrence of R⁸ is —OCH₃.

In an exemplary embodiment, R³ and R⁴ are each isopropyl, R² and R⁶ areeach methyl, R⁵ is —H, R⁷ is sec-butyl, each occurrence of R⁸ is —OCH₃,and R⁹ is —H.

In one embodiment, Z is —O— or —NH—.

In one embodiment, R¹⁰ is aryl.

In an exemplary embodiment, R¹⁰ is -phenyl.

In an exemplary embodiment, when Z is —O—, R¹¹ is —H, methyl or t-butyl.

In one embodiment, when Z is —NH, R¹¹ is —CH(R¹⁵)₂, wherein R¹⁵ is—(CH₂)_(n)—N(R¹⁶)₂, and R¹⁶ is —C₁-C₈ alkyl or —(CH₂)_(n)—COOH.

In another embodiment, when Z is —NH, R¹¹ is —CH(R¹⁵)₂, wherein R¹⁵ is—(CH₂)_(n)—SO₃H.

One exemplary auristatin embodiment of formula V is MMAE, wherein thewavy line indicates the covalent attachment to a L, L′, V, V′, Y, Y′,(RR′), R′ or (C)_(n) group of a compound of the invention (e.g. acompound of Formula I, II or IV):

An exemplary auristatin embodiment of formula VI is MMAF, wherein thewavy line indicates the covalent attachment to a linker (L) of anantibody-drug conjugate (see US 2005/0238649 and Doronina et al. (2006)Bioconjugate Cfiem. 17: 114-124):

Other exemplary Z embodiments include monomethylvaline compounds havingphenylalanine carboxy modifications at the C-terminus of thepentapeptide auristatin drug moiety (WO 2007/008848) andmonomethylvaline compounds having phenylalanine sidechain modificationsat the C-terminus of the pentapeptide auristatin drug moiety (WO2007/008603).

Other drug moieties include the following MMAF derivatives, wherein thewavy line indicates the covalent attachment to a L, L′, V, V′, Y, Y′,(RR′), R′ or (C)_(n) group of a compound of the invention (e.g. acompound of Formula I, II or IV)::

An example of a linker of the invention comprising a a lysine residue as(C)_(n) moiety, a valine-citrulline as the (V) moiety, a PAB as the (Y)moiety together with a MMAF as the (Z) moiety is shown below(corresponding to compound Ia-1):

In one embodiment, the Z moiety is an epothilone or epothilonederivative. An epothilone is a cyclic molecule with a 16-membered ringand variable substituents and pharmaceutical activity as a cytostaticagent that binds to tubulin Various epothilone derivatives are known,including variants with 14-, 15- or 18-membered rings have also beendeveloped (e.g. WO2011085523; WO2009105969). Examples of epothilones orepothilone analogs or derivative in the context of the present inventioninclude epothilone A, epothilone B, epothilone C₁₋₁₃-alkyl-epothilone Cderivatives, epothilone D, trans-epothilone D, epothilone E, epothiloneF, an effector conjugate of epothilone, Sagopilone, or any of theepothilones referred to in the literature as ixabepilone (BMS-247550),BMS-310705, EPO-906, Patupilone, Kos-862, Kos-1584, Kos-1803 and ABJ879, and pharmaceutically active salts thereof. The production ofepothilones, their precursors and derivatives is generally carried outaccording to the methods known to one skilled in the art. Suitablemethods are, for example, described in DE 19907588, WO 98/25929, WO99/58534, WO 99/2514, WO 99/67252, WO 99/67253, WO 99/7692, EP 99/4915,WO 00/485, WO 00/1333, WO 00/66589, WO 00/49019, WO 00/49020, WO00/49021, WO 00/71521, WO 00/37473, WO 00/57874, WO 01/92255, WO01/81342, WO 01/73103, WO 01/64650, WO 01/70716, U.S. Pat. No.6,204,388, U.S. Pat. No. 6,387,927, U.S. Pat. No. 6,380,394, U.S. Ser.No. 02/52,028, U.S. Ser. No. 02/58,286, U.S. Ser. No. 02/62,030, WO02/32844, WO 02/30356, WO 02/32844, WO 02/14323, and WO 02/8440. Furtherepothilones are described in WO 93/10102, WO 98/25929, WO 99/02514, WO99/07692, WO 99/02514, WO 99/67252, WO 00/49021, WO 00/66589, WO00/71521, WO 01/027308, WO 02/080846, WO 03/074053, WO 2004/014919.

Other useful therapeutics are set forth in the Physician's DeskReference and in the Orange Book maintained by the US Food and DrugAdministration (FDA). New drugs are continually being discovered anddeveloped, and the present invention provides that these new drugs mayalso be incorporated into a compound of this invention.

Chelated metals include chelates of di- or tripositive metals having acoordination number from 2 to 8 inclusive. Particular examples of suchmetals include technetium (Tc), rhenium (Re), cobalt (Co), copper (Cu),gold (Au), silver (Ag), lead (Pb), bismuth (Bi), indium (In), gallium(Ga), yttrium (Y), terbium (Tb), gadolinium (Gd), and scandium (Sc). Ingeneral the metal is preferably a radionuclide. Particular radionuclidesinclude ^(99m)Tc, ¹⁸⁶Re, ¹⁸⁸Re, ⁵⁸Co, ⁶⁰Co, ⁶⁷Cu, ¹⁹⁵Au, ¹⁹⁹Au, ¹¹⁰Ag,²⁰³Pb, ²⁰⁶Bi, ²⁰⁷Bi, ¹¹¹In, ⁶⁷Ga, ⁸⁸Y, ⁹⁰Y, ¹⁶⁰Tb, ¹⁵³Gd and ⁴⁷Sc.

The chelated metal may be for example one of the above types of metalchelated with any suitable polydentate chelating agent, for exampleacyclic or cyclic polyamines, polyethers, (e.g. crown ethers andderivatives thereof); polyamides; porphyrins; and carbocyclicderivatives.

In general, the type of chelating agent will depend on the metal in use.One particularly useful group of chelating agents in conjugatesaccording to the invention, however, are acyclic and cyclic polyamines,especially polyaminocarboxylic acids, for examplediethylenetriaminepentaacetic acid and derivatives thereof, andmacrocyclic amines, e.g. cyclic tri-aza and tetra-aza derivatives (forexample as described in PCT publication no. WO 92/22583); andpolyamides, especially desferriox-amine and derivatives thereof.

Other effector molecules may include detectable substances useful forexample in diagnosis. Examples of detectable substances include variousenzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, radioactive nuclides, positronemitting metals (for use in positron emission tomography), andnonradioactive paramagnetic metal ions. See generally U.S. Pat. No.4,741,900 for metal ions which can be conjugated to antibodies for useas diagnostics. Suitable enzymes include horseradish peroxidase,alkaline phosphatase, beta-galactosidase, or acetylcholinesterase;suitable prosthetic groups include streptavidin, avidin and biotin;suitable fluorescent materials include umbelliferone, fluorescein,fluorescein isothiocyanate, rhodamine, dichlorotriazinylaminefluorescein, dansyl chloride and phycoerytbrin; suitable luminescentmaterials include luminol; suitable bioluminescent materials includeluciferase, luciferin, and aequorin; and suitable radioactive nuclidesinclude 125I, ¹³¹I, ¹¹¹In and ⁹⁹Tc.

Synthetic or naturally occurring polymers for use as effector moleculesinclude, for example optionally substituted straight or branched chainpolyalkylene, polyalkenylene, or polyoxyalkylene polymers or branched orunbranched polysaccharides, e.g. a homo- or hetero-polysaccharide suchas lactose, amylose, dextran or glycogen.

Particular optional substituents which may be present on theabove-mentioned synthetic polymers include one or more hydroxy, methylor methoxy groups. Particular examples of synthetic polymers includeoptionally substituted straight or branched chain poly(ethyleneglycol),poly(propyleneglycol), poly(vinylalcohol) or derivatives thereof,especially optionally substituted poly(ethyleneglycol) such asmethoxypoly(ethyleneglycol) or derivatives thereof. Such compounds, whenused as a moiety Z can be employed as a moiety that improves thepharmacokinetic properties of the antibody.

The size of the polymer may be varied as desired, but will generally bein an average molecular weight range from 500 Da to 50,000 Da,preferably from 5,000 to 40,000 Da and more preferably from 10,000 to40,000 Da and 20,000 to 40,000 Da. The polymer size may in particular beselected on the basis of the intended use of the product for exampleability to localize to certain tissues such as tumors or extendcirculating half-life (for review see Chapman, 2002, Advanced DrugDelivery Reviews, 54, 531-545). Thus, for example, where the product isintended to leave the circulation and penetrate tissue, for example foruse in the treatment of a tumor, it may be advantageous to use a smallmolecular weight polymer, for example with a molecular weight of around5,000 Da. For applications where the product remains in the circulation,it may be advantageous to use a higher molecular weight polymer, forexample having a molecular weight in the range from 20,000 Da to 40,000Da.

Particularly preferred polymers include a polyalkylene polymer, such asa poly(ethyleneglycol) or, especially, a methoxypoly(ethyleneglycol) ora derivative thereof, and especially with a molecular weight in therange from about 10,000 Da to about 40,000 Da.

In another embodiment, z′ equals 1, each V, Y or V—Y (including whetherany V and Y is a V′ or Y′) moiety contains a single attachment site fora functional group of Z.

In another embodiment, a one V (or V′), Y, (or Y′) or V—Y (or V′—Y′,V—Y′) moiety is attached to more than one Z moiety via multiplefunctional groups R on the said V, Y or V—Y moiety. Optionally, the oneor more V (or V′) moieties comprise a polymer, optionally anoligoethylene glycol or a polyethylene glycol or a derivative thereof.

Any one of the Z moieties disclosed herein can be utilized in FormulaIa, IIII, and IVa. Any one of the Z moieties described herein can beused in combination with any of the (C)_(n), X, L, V, R, Y, Z, M, z, q,and r groups described herein. Any one of the Z moieties describedherein can be used in combination with any of the R′, L′, V′, Y′, z′,q′, and r′ groups described herein.

Antibody-Z Conjugates

In one embodiment, a linking reagent (e.g. of Formula Ia) is directlyconjugated to an antibody or antibody fragment, without requirement fora step of reaction involving reactive groups R and R′. In one aspect, anantibody or antibody fragment of the invention comprises afunctionalized glutamine residue of Formula IVa, below.

(Q)-NH—(C)_(n)—X-L-(V—(Y—(Z)_(z))_(q))_(r)  Formula IVa

or a pharmaceutically acceptable salt thereof;

wherein:

Q is glutamine residue present in an antibody or antibody fragment;

(C)_(n) is a substituted or unsubstituted alkyl or heteroalkyl chain,wherein any carbon of the chain is optionally substituted with analkoxy, hydroxyl, alkylcarbonyloxy, alkyl-S—, thiol, alkyl-C(O)S—,amine, alkylamine, amide, or alkylamide (e.g. a O, N or S atom of anether, ester, thioether, thioester, amine, alkylamine, amide, oralkylamide);

n is an integer selected from among the range of 2 to 20;

X is NH, O, S, or absent;

L is a bond or a carbon comprising framework, preferably of 1 to 200atoms substituted at one or more atoms, optionally wherein the carboncomprising framework is a linear hydrocarbon, a symmetrically orasymmetrically branched hydrocarbon monosaccharide, disaccharide, linearor branched oligosaccharide (asymmetrically branched or symmetricallybranched), other natural linear or branched oligomers (asymmetricallybranched or symmetrically branched), or a dimer, trimer, or higheroligomer (linear, asymmetrically branched or symmetrically branched)resulting from any chain-growth or step-growth polymerization process;

r is an integer selected among 1, 2, 3 or 4;

q is an integer selected among 1, 2, 3 or 4;

z is an integer selected among 1, 2, 3 or 4; and

V is independently absent, a non-cleavable moiety or aconditionally-cleavable moiety that can optionally be cleaved ortransformed by a chemical, photochemical, physical, biological, orenzymatic process (e.g. cleavage of V ultimately leading to release ofone or more moieties subsequently or ultimately linked to V, for examplea Z moiety). In some embodiments, V is, preferably, a di-, tri-, tetra-,or oligopeptide as described below in the section entitled “The VMoiety”;

Y is independently absent or a spacer (e.g., a self-eliminating spacersystem or a non-self-elimination spacer system) which is comprised of 1or more spacers; and

Z is a moiety-of-interest, optionally a moiety that improves thepharmacokinetic properties, or a therapeutic moiety or a diagnosticmoiety. Preferably, Z is a cytotoxic anti-cancer agent, e.g. a compoundselected from the group consisting of taxanes, anthracyclines,camptothecins, epothilones, mytomycins, combretastatins, vincaalkaloids, nitrogen mustards, maytansinoids, calicheamycins,duocarmycins, tubulysins, amatoxins, dolastatins and auristatins,enediynes, radioisotopes, therapeutic proteins and peptides, and toxinsor fragments thereof.

Generally, each Z is directly coupled to either Y or V when Y is absent,or L when both Y and V are absent.

It will be appreciated that Formula IVa can for convenience also beexpressed as (Ab)—NH—(C)_(n)—X-L-(V—(Y—(Z)_(z))_(q))_(r) (Formula IVa),where (Ab) is an immunoglobulin (Ab) is conjugated via a glutamine (Q)residue to an NH of the linking reagent (e.g the compound of FormulaIa).

Examples of antibodies or antibody fragments of Formula IVa include butare not limited to antibodies and fragments attached via an amide bond(e.g. through an acceptor glutamine residue in the primary sequence ofthe antibody or antibody fragment) to a compound selected from the groupconsisting of compounds Ia-1 to Ia-23 (wherein the terminal NH₂— of eachof said compound Ia-1 to Ia-23 is replaced by a moiety ((Q)-NH—) whenattached to the antibody or fragment, wherein Q is glutamine residuepresent in an antibody or antibody fragment.

The antibody conjugates resulting from the reaction of the compounds ofFormula Ib or III with an antibody conjugated to a lysine-based linkerwill yield an antibody conjugate in which a moiety Z is connected tolinker L (or L′) when Y (or Y′) and V (or V′) are absent, to the spacersystem Y (or Y′) or, when Y (or Y′) is absent, to V (or V). Optionallysaid connections are via linking group (RR′) of M.

The conjugates resulting from the reaction yield an antibody (Ab) whichis conjugated (i.e., covalently attached) via an acceptor glutamineresidue (Q) present on the antibody to a NH group of a lysine-basedlinker, and one or more moieties (Z) through optional linking group(RR′), optional linker (V or V′) and/or optional spacer (Y or Y′).

In one embodiment, the (RR′) remains present in a conjugated antibody orantibody fragment, in which case a Formula IV will comprise an (M)moiety. Such an antibody or antibody fragment of the invention comprisesa functionalized glutamine residue of Formula IVb, below,

(Q)-NH—(C)_(n)—X-L-(V—(Y-(M)_(z))_(q))_(r)  Formula IVb

or a pharmaceutically acceptable salt or solvate thereof;

wherein:

Q is glutamine residue present in an antibody or antibody fragment;

(C)_(n) is a substituted or unsubstituted alkyl or heteroalkyl chain,wherein any carbon of the chain is optionally substituted with analkoxy, hydroxyl, alkylcarbonyloxy, alkyl-S—, thiol, alkyl-C(O)S—,amine, alkylamine, amide, or alkylamide;

n is an integer selected from among the range of 2 to 20;

X is NH, O, S, or absent;

L is a bond or a carbon comprising framework, preferably of 1 to 200atoms substituted at one or more atoms, optionally wherein the carboncomprising framework is a linear hydrocarbon, a symmetrically orasymmetrically branched hydrocarbon monosaccharide, disaccharide, linearor branched oligosaccharide (asymmetrically branched or symmetricallybranched), other natural linear or branched oligomers (asymmetricallybranched or symmetrically branched), or a dimer, trimer, or higheroligomer (linear, asymmetrically branched or symmetrically branched)resulting from any chain-growth or step-growth polymerization process;

r is an integer selected among 1, 2, 3 or 4;

q is an integer selected among 1, 2, 3 or 4;

z is an integer selected among 1, 2, 3 or 4; and

V is independently absent, a non-cleavable moiety or aconditionally-cleavable moiety that can optionally be cleaved ortransformed by a chemical, photochemical, physical, biological, orenzymatic process (e.g. cleavage of V ultimately leading to release ofone or more moieties subsequently or ultimately linked to V, for examplea Z moiety). In some embodiments, V is, preferably, a di-, tri-, tetra-,or oligopeptide as described below in the section entitled “The VMoiety”;

Y is independently absent or a spacer (e.g., a self-eliminating spacersystem or a non-self-elimination spacer system) which is comprised of 1or more spacers; and

M is independently: R or (RR′)-L′-(V′—(Y′—(Z)_(r′))_(q′))_(r′), whereineach of L′, V′, Y′, z′, q′, and r′ are as defined in Formula III (or aredefined as L, V, Y, z, q and r, respectively,

Z is a moiety-of-interest, optionally a moiety that improves thepharmacokinetic properties, or a therapeutic moiety or a diagnosticmoiety, R is as defined in Formula I and wherein each (RR′) is anaddition product between an R of Formula I and its complementary R′ ofFormula III (see, for example, FIG. 1 and FIG. 2).

Thus, RR′ can be for example an addition product of a thio-maleimide (orhaloacetamide) addition, for example, aN,S-disubstituted-3-thio-pyrrolidine-2,5-dione; Staudinger ligation, forexample, a N,3- or N,4-substitued-5-dipenylphosphinoxide-benzoic amide;Huisgen 1,3-cycloaddition (click reaction), for example, aN,S-disubstituted-3-thio-pyrrolidine-2,5-dione,1,4-disubstituted-1,2,3-triazole, 3,5-disubstituted-isooxazole, or3,5-disubstituted-tetrazole; Diels-Alder cycloaddition adduct, forexample the 2,4-cycloaddition product between an O orN-substituted-5-norbornene-2-carboxylic ester or amide,N-substituted-5-norbornene-2,3-dicarboxylic imide, O orN-substituted-7-oxonorbornene-5-carboxylic ester or amide, orN-substituted-7-oxonorbornene-5,6-dicarboxylic imide and a 9-substitutedanthracene or 3-substituted 1,2,4,5-tetrazine; or any high yieldselective amidation or imidization reaction. Some reactions and thecorresponding RR′ reaction products are illustrated in FIGS. 1 and 2.

Examples of RR′ include:

wherein (*) indicates the site of attachment of —(C)_(n), X, L, L′, V,V′, Y, Y′ or Z. RR′ can be in either orientation with respect to theirattachment to —(C)_(n) X, L, L′, V, V′, Y, Y′ or Z).

Optionally, the antibody conjugate comprises a group (RR′) representingthe remainder of a reactive moiety R when R has reacted with a reactivemoiety R′, wherein the group (RR′) connects (a) an L to a Z, a V or a Y,(b) a V to a Z or a Y, or (c) a Y to a Z. For example, any V, Y and/or Zmay be characterized as comprising a (RR′) group. Any L, V, Y may be anL′, V′ or Y′, respectively.

It will be appreciated that Formula IVb can for convenience also beexpressed as (Ab)—NH—(C)_(n)—X-L-(V—(Y-(M)_(z))_(q))_(r), where (Ab) isan immunoglobulin (Ab) is conjugated via a glutamine (Q) residue to anNH of the linking reagent (e.g the compound of Formula Ib or Ic).

Examples of antibodies or antibody fragments of Formula IVb include butare not limited to:

In one embodiment, the glutamine (Q) is present in the constant regionof an antibody heavy chain. In one embodiment, the glutamine (Q) is atposition 295. In one embodiment, an acceptor glutamine (Q) is atposition 297 (e.g., a N297Q substitution). In one embodiment, theantibody comprises a substitution of an asparagine at position 297 witha non-asparagine, non-aspartic acid, non-glutamine, residue.

In one embodiment, a single surface exposed acceptor glutamine (Q) ispresent in the constant region of an antibody heavy chain. Optionallythe antibody optionally comprises two heavy chains; such an antibodywill comprise two functionalized acceptor glutamines of Formula IV perantibody molecule. Optionally said single acceptor glutamine (Q) islocated at position 295. In one embodiment, the antibody comprises aN297Q substitution such that said single glutamine (Q) is located atposition 295. In one embodiment, the antibody comprises a Q295substitution (the glutamine at residue 295 is substituted by anon-glutamine residue) and a N297Q substitution, and said singleglutamine (Q) is located at position 297.

In one embodiment, two surface exposed acceptor glutamines (Q) arepresent in the constant region of an antibody heavy chain Optionally theantibody optionally comprises two heavy chains; such an antibody willcomprise four functionalized acceptor glutamines of Formula IV perantibody molecule. Optionally the first glutamine (Q) is located atposition 295 and the second glutamine (Q) is located at position 297(e.g, a N297Q substitution).

Exemplary Methods for Preparing Compounds and Antibody-Conjugates

A general scheme for preparing conjugated antibodies is shown in FIG.10. Exemplary compounds can be prepared using known synthesis methodsand starting reagents. In the examples below, all chemicals arepurchased from Sigma-Aldrich, Fluka or Pierce Thermo scientific unlessotherwise stated. All chemicals and solvents are used without furtherpurification. Reactions are monitored by HPLC or by thin layerchromatography (TLC) using precoated silica gel 60 F aluminum sheets(Merck), and visualized by UV absorption or stained.

1. N-succinimidyl-5-acetylthioesters as Building Blocks

In a first step, mono-Boc-protected cadaverin is reacted withN-succinimidyl-S-acetylthioacetate (SATA) or succinimidylacetyl(thiotetraethyleneglycol (SAT(PEG)₄ orN-succinimidyl-5-acetylthiopropionate (SATP) to give the correspondingintermediates S-acetyl-cadaverin-Boc. Boc-deprotection is achieved inacidic conditions to give S-acetyl-cadaverin. Purification is achievedusing reversed phase high performance liquid chromatography (RP-HPLC) togive the final product. The reaction scheme is shown in FIG. 3.

The antibody conjugate is then prepared as shown in FIG. 11 (“R” is amoiety-of-interest Z). Chimeric antibody chCE7 (1 mg) in PBS buffer (0.1mol/L NaCl and 0.05 mol/L sodium phosphate buffer, pH 7.4) are incubatedwith 100 units (0.2 μL) of N-glycosidase F (PNGase F) fromFlavobacterium meningosepticum (New England BioLabs, Ipswich, UK) at 37°C. overnight to deglycosylate the antibody. The enzyme is then removedby centrifugation-dialysis (Vivaspin MWCO 50 kDa, Vivascience, Winkel,Switzerland). The product can be analyzed by LC/MS.

IgG antibody chCE7 is reacted with S-acetyl-cadaverin in the presence ofrecombinant BTG (EC 2.3.2.13) from streptomyces mobaraensis (Zedira,Darmstadt, Germany) at a concentration of 1-20 U/mL in potassium-freephosphate buffered saline (PBS; pH 8) at 37° C. After 4 h to severaldays (depending on the antibody and the ligand), steady-state conditionsare achieved. Excess ligand and enzyme are then removed usingcentrifugation-dialysis (Vivaspin MWCO 50 kDa, Vivascience, Winkel,Switzerland). Reactions are monitored by LC/MS.

De-protection (deacylation) of the S-acetyl-protected IgG to generate afree sulfhydryl is accomplished using hydroxylamine-HCl. Then, theantibody-lysine-based linker conjugate is added to maleimide (orhaloacetamide, e.g., bromoacetamide) containing compound at 4° C. for 1h and the conjugation reaction is quenched by adding a 20-fold excess ofcysteine. The reaction mixture is concentrated by centrifugalultrafiltration and buffer-exchanged through Sephadex G-25 equilibratedwith PBS at 4° C. The conjugate is then sterile filtered through a 0.2μm filter.

2. Azide Moieties

Mono-Boc-protected cadaverin is reacted with N-hydroxysuccinimide esterethane azide (NHS-azide) or N-hydroxysuccinimide estertetraoxapentadecane azide (NHS-PEG4-Azide) or N-hydroxysuccinimide esterdodecaoxanonatriacontane azide (NHS-PEG12-Azide) to give theintermediate azide-cadaverin-Boc. Boc-deprotection is achieved thepresence of trifluoroacetic acid (TFA). Purification using RP-HPLCprovide azide-cadaverin. The reaction scheme is shown in FIG. 5.

The Antibody conjugate is then prepared as shown in FIG. 12 (“R” is amoiety-of-interest Z). Chimeric antibody chCE7 is deglycosylated andazide-cadaverin conjugates are prepared. The azide-modified antibody isreacted with the alkyne-reactive moiety compound using standardconditions for click chemistry.

3. Norbornene Moiety

In the first step, norbornene carboxylic acid is activated to asulfo-NHS ester in the presence of1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). Then,mono-Boc-protected cadaverin is added and reacted with the reactiveester to give norbornyl-cadaverin-Boc. Deprotection of Boc is achievedby acidic treatment. Purification using RP-HPLC providenorbornyl-cadaverin. The reaction scheme is shown in FIG. 8.

The Antibody conjugate is then prepared as shown in FIG. 13 (“R” is amoiety-of-interest Z). Chimeric antibody chCE7 is deglycosylated andazide-cadaverin conjugates are prepared. The norbornene-modifiedantibody (in PBS pH 7.4) is reacted with a molar excess of tetrazinereactive moiety compound (in DMSO or appropriate organic solvent) (molarexcess is calculated based on initial norbornene reactionstoichiometry). The reaction is incubated at RT for 5 h and subsequentlypurified using centrifugal filtration to yield the completed antibodyconjugate.

4. Alkyne Moiety

H-Lys-(Boc)-OMe is alkylated with propargyl glycine in the presence ofCs. Boc deprotection is achieved in a mixture of TFA (10%) anddichloromethane (DCM). Deprotection of the methyl ester with aqueousNaOH gives the glycan-lysine derivative in 50% yield. The reactionscheme is shown in FIG. 7.

The Antibody conjugate is then prepared as shown in FIG. 14 (“R” is amoiety-of-interest Z). Chimeric antibody chCE7 is deglycosylated andazide-cadaverin conjugates are prepared. The alkyne-modified antibody isreacted with the azide-reactive moiety compound using standardconditions for click chemistry. For conjugation by Staudinger ligation,the azido-derivated antibody is mixed with the phosphine-moiety compoundand incubate at 37° C. for 2-4 hours, or at room temperature or 4° C.incubation during 16-24 hours. (final concentration of the antibody ispreferably >2 mg/mL).

Uses of Compounds

In one aspect, this invention relates to use of a compound of Formula Ifor the preparation of an antibody conjugate of Formula II.

In another aspect, this invention relates to use of a compound ofFormula III and/or an antibody conjugate of Formula II, for thepreparation of an antibody conjugate of Formula IV.

In yet another aspect, the invention relates to the use of any of thecompounds of the invention for the manufacture of a diagnostic product,a kit and/or a pharmaceutical preparation for the treatment or diagnosisof a mammal in need thereof. In one embodiment, the invention relates tothe use of any of the compounds defined above for the manufacture of apharmaceutical composition for the treatment of a tumor or infectiousdisease in a mammal

Also the invention relates to any of the compounds defined above as amedicament or an active component or active substance in a medicament Ina further aspect the invention relates to a method for preparing apharmaceutical composition containing a compound as defined above, toprovide a solid or a liquid formulation for administration orally,topically, or by injection. Such a method or process at least comprisesthe step of mixing the compound with a pharmaceutically acceptablecarrier.

In one aspect, this invention relates to a method to affect or prevent apredefined condition by exerting a certain effect, or detect a certaincondition using a compound of the present invention, or a(pharmaceutical) composition comprising a compound of this invention.

In one embodiment, this invention relates to a method of detecting thepresence of a certain condition, e.g., the presence of an enzyme, thepresence of a certain pH, the presence of a (bio)molecule, the presenceof a substrate, or the presence of a certain oxygen concentration, witha compound of this invention, either in vivo or ex vivo.

In one embodiment, this invention relates to a method of determining anenzyme ex vivo, e.g., in a diagnostic assay, using a compound of thisinvention by incubating a sample (possibly) containing said enzyme witha compound of this invention containing one or more diagnostic moietiesZ and a substrate for said (proteolytic) enzyme, and observing releaseof said Z moieties. The phrase “determining an enzyme” means bothqualitative analysis, i.e., detecting the presence of the enzyme,determining whether it is present, and quantitative analysis, i.e.,quantifying the enzyme, determining the enzyme activity present in thesample. An enzyme can also be indirectly determined via its pro-enzymecontaining a recognition site, e.g., an activation site, cleavable bysaid enzyme to be determined. Cleavage of the pro-enzyme can in suchcase be detected by observing the resulting activity using a suitablecompound of the present invention.

In one embodiment the invention relates to a diagnostic assay method (invivo or ex vivo) in which a compound according to the invention is used.

In a further embodiment the invention relates to a method in which thepresence or amount of an enzyme is determined by using a compoundaccording to the invention.

In one embodiment, this invention relates to a method to affect orprevent a predefined condition, e.g., a disease such as an autoimmunedisease, a microbial disease, or cancer, by exerting an effect using acompound of this invention.

In a further embodiment, the invention relates to a method of treating amammal being in need thereof, whereby the method comprises theadministration of a pharmaceutical composition to the mammal in atherapeutically effective dose.

In a further embodiment, this invention relates to a method of treatinga mammal having an illness characterized by undesired (cell)proliferation with a compound of this invention. In another embodimentthis invention relates to a method of treating a mammal carrying a tumorwith a compound of this invention. In yet another embodiment thisinvention relates to a method of treating a mammal having aninflammatory disease with a compound of this invention. In yet anotherembodiment this invention relates to a method of treating a mammalhaving an autoimmune disease with a compound of this invention. In yetanother embodiment this invention relates to a method of treating amammal having a bacterial or microbial infection with a compound of thisinvention.

In one embodiment, the invention relates to a method of treating cancerin a mammal, whereby the method comprises the administration of apharmaceutical composition to the mammal in a therapeutically effectivedose.

In one embodiment, a compound of the invention is used to treat anillness characterized by undesired proliferation. In another embodiment,a compound of the invention is used to treat an illness characterized byundesired (cell) proliferation. In another embodiment, a compound of theinvention is used to treat a tumor. In yet another embodiment, acompound of the invention is used to treat an inflammatory disease. Inyet another embodiment a compound of the invention is used to treat anautoimmune disease. In yet another embodiment a compound of theinvention is used to treat a bacterial or microbial infection.

In one embodiment, the compound of the invention is capable of beinginternalized into cells that express an antigen to which the antibodybinds (e.g. a tumor or viral antigen) and/or induces internalization ofthe antigen on said antigen-expressing cells. In one embodiment, thecompound of the invention is toxic to a cell upon internalization (i.e.the compound comprises a moiety Z that is toxic to a cell). Preferablysuch compounds can be used in methods of killing or eliminating cells,preferably wherein said cells are tumor cells.

The invention also relates to pharmaceutical compositions comprising thecompounds of the invention as defined above a compound of the inventionmay be administered in purified form together with a pharmaceuticalcarrier as a pharmaceutical composition. The preferred form depends onthe intended mode of administration and therapeutic or diagnosticapplication. The pharmaceutical carrier can be any compatible, nontoxicsubstance suitable to deliver the compounds of the invention to thepatient. Pharmaceutically acceptable carriers are well known m the artand include, for example, aqueous solutions such as (sterile) water orphysiologically buffered saline or other solvents or vehicles such asglycols, glycerol, oils such as olive oil or injectable organic esters,alcohol, fats, waxes, and inert solids A pharmaceutically acceptablecarrier may further contain physiologically acceptable compounds thatact for example to stabilize or to increase the absorption of thecompounds of the invention. Such physiologically acceptable compoundsinclude, for example, carbohydrates, such as glucose, sucrose ordextrans, antioxidants, such as ascorbic acid or glutathione, chelatingagents, low molecular weight proteins or other stabilizers orexcipients. One skilled in the art would know that the choice of apharmaceutically acceptable carrier, including a physiologicallyacceptable compound, depends, for example, on the route ofadministration of the composition Pharmaceutically acceptable adjuvants,buffering agents, dispersing agents, and the like, may also beincorporated into the pharmaceutical compositions.

For oral administration, the active ingredient can be administered insolid dosage forms, such as capsules, tablets, and powders, or m liquiddosage forms, such as elixirs, syrups, and suspensions. Activecomponent(s) can be encapsulated in gelatin capsules together withinactive ingredients and powdered carriers, such as glucose, lactose,sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesiumstearate, stearic acid, sodium saccharin, talcum, magnesium carbonateand the like. Examples of additional inactive ingredients that may beadded to provide desirable color, taste, stability, buffering capacity,dispersion or other known desirable features are red iron oxide, silicagel, sodium lauryl sulfate, titanium dioxide, edible white ink and thelike. Similar diluents can be used to make compressed tablets. Bothtablets and capsules can be manufactured as sustained release productsto provide for continuous release of medication over a period of hours.Compressed tablets can be sugar-coated or film-coated to mask anyunpleasant taste and protect the tablet from the atmosphere, orenteric-coated for selective disintegration in the gastrointestinaltract Liquid dosage forms for oral administration can contain coloringand flavoring to increase patient acceptance.

The compounds of the invention are however preferably administeredparenterally. Preparations of the compounds of the invention forparenteral administration must be sterile Sterilization is readilyaccomplished by filtration through sterile filtration membranes,optionally prior to or following lyophilization and reconstitution. Theparenteral route for administration of compounds of the invention is inaccord with known methods, e.g. injection or infusion by intravenous,intraperitoneal, intramuscular, intraarterial, or intralesional routes.The compounds of the invention may be administered continuously byinfusion or by bolus injection. A typical composition for intravenousinfusion could be made up to contain 100 to 500 ml of sterile 0.9% NaClor 5% glucose optionally supplemented with a 20% albumin solution and 1mg to 10 g of the compound of the invention, depending on the particulartype of compound of the invention and its required dosing regime.Methods for preparing parenterally administrable compositions are wellknown in the art.

EXAMPLES Materials and Methods Antibodies

chADC1 (or chimADC1) is an antibody specific for a human tumor antigen,chimADC1 (or chADC1), a chimeric antibody generated in mice andconverted to human IgG1 isotype. chCE7 is specific for human L1-CAM andis composed of murine VL and murine VH fused to the Fc part of humanIgG1 (see, e.g., Jeger et al., (2010) Angew. Chem. Int., 49, 9995-9997and Knogler et al, (2007) Clin Caner res., 13, 603-611). SGN-35 isspecific for human CD30 and is described in Maeda et al. 2010 CancerSci. 101(1):224-230 and U.S. Pat. No. 7,090,843. ChADC1, SGN-35 andchCE7 are full length tetrameric antibodies with one acceptor glutamineper heavy chain at amino acid residue 295 (Kabat EU), i.e. a total oftwo acceptor glutamines. Unless otherwise indicated, chADC1, SGN-35 andchCE7 antibodies without the Fc mutations used in BTG coupling reactionwere deglycosylated with PNGase F.

Fc Mutant Antibodies.

Variants of antibodies chimADC1 and SGN-35 were constructed thatcontained a N297S mutation; this antibody thus had one acceptorglutamine per heavy chain at amino acid residues 295 (Kabat EU), i.e. atotal of two acceptor glutamines per tetrameric antibody, and wereaglycosylated.

Variants of antibodies chimADC1, SGN-35 and chCE7 were also constructedthat contained a N297Q mutation; these antibodies thus had two acceptorglutamine per heavy chain at amino acid residues 295 and 297 (Kabat EU),i.e. a total of four acceptor glutamines, and were aglycosylated. Afurther variant of chCE7 contained both Q295N and N297Q mutations.

For chimADC1 and SGN-35, two different sequences having the N297S orN297Q mutations in the human constant region of γ1 antibodies weresynthetized by MWG-Biotech. These two mutated sequences were designedrespectively N297S and N297Q.

The nucleic acid and amino acid sequences synthesized for the N297Sconstruct (the mutation is underlined) is shown below:

(SEQ ID NO:5)GGGCCCAAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTGGAGCCCAAGAGCTGTGACAAGACCCACACCTGCCCCCCCTGCCCAGCCCCCGAGCTGCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGTGTGGTGGTGGACGTGTCCCACGAGGACCCAGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTAC AGCAGCACCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCAGCCCCAATCGAAAAGACCATCAGCAAGGCCAAGGGCCAGCCAAGAGAGCCCCAGGTGTACACCCTGCCACCCAGCAGGGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCAAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCAGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCCAGGCAAGTGATGAATTC (SEQ ID NO:6)G P S V F P L A P S S K S T S G G T A A L G C L V K D Y F P E P V T V S W NS G A L T S G V H T F P A V L Q S S G L Y S L S S V V T V P S S S L G T Q TY I C N V N H K P S N T K V D K R V E P K S C D K T H T C P P C P A P E L LG G P S V F L F P P K P K D T L M I S R T P E V T C V V V D V S H E D P E VK F N W Y V D G V E V H N A K T K P R E E Q Y S S T Y R V V S V L T V L H QD W L N G K E Y K C K V S N K A L P A P I E K T I S K A K G Q P R E P Q V YT L P P S R E E M T K N Q V S L T C L V K G F Y P S D I A V E W E S N G Q PE N N Y K T T P P V L D S D G S F F L Y S K L T V D K S R W Q Q G N V F S CS V M H E A L H N H Y T Q K S L S L S P G K 

The nucleic acid and amino acid sequences synthesized for the N297Qconstruct (the mutation is underlined) is shown below:

(SEQ ID NO:7)GGGCCCAAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTGGAGCCCAAGAGCTGTGACAAGACCCACACCTGCCCCCCCTGCCCAGCCCCCGAGCTGCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGTGTGGTGGTGGACGTGTCCCACGAGGACCCAGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTACCAAAGCACCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCAGCCCCAATCGAAAAGACCATCAGCAAGGCCAAGGGCCAGCCAAGAGAGCCCCAGGTGTACACCCTGCCACCCAGCAGGGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCAAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCAGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCCAGGCAAGTGATGAATTC (SEQ ID NO: 8)G P S V F P L A P S S K S T S G G T A A L G C L V K D Y F P E P V T V S W NS G A L T S G V H T F P A V L Q S S G L Y S L S S V V T V P S S S L G T Q TY I C N V N H K P S N T K V D K R V E P K S C D K T H T C P P C P A P E L LG G P S V F L F P P K P K D T L M I S R T P E V T C V V V D V S H E D P E VK F N W Y V D G V E V H N A K T K P R E E Q Y Q S T Y R V V S V L T V L H QD W L N G K E Y K C K V S N K A L P A P I E K T I S K A K G Q P R E P Q V YT L P P S R E E M T K N Q V S L T C L V K G F Y P S D I A V E W E S N G Q PE N N Y K T T P P V L D S D G S F F L Y S K L T V D K S R W Q Q G N V F S CS V M H E A L H N H Y T Q K S L S L S P G K

These sequences were then digested from the MWG-Biotech cloning vectorwith the Apal and EcoRI restriction enzymes and cloned into the vector Bdigested with the same restriction enzymes (B N297S and B N297Q). Lightchain and heavy chain of the variable domains of the chADC1 antibodywere amplified by PCR and the purified products of the PCR were clonedtogether into the vectors B N297S and N297Q using the InFusion cloningsystem (Ozyme) to create bicistronic vectors. The bicistronic vectorsgenerated were then sequenced and validated prior to cell transfection.CHO cells were transfected with the vectors encoding chADC1 or SGN-30N297S and N297Q and cells were grown in rolling bottle to produce largequantities of antibodies that were purified from the harvestedsupernatant.

For chCE7 (anti-L1-CAM antibody), cDNAs from heavy and light chain werecloned separately into the HindIII/BamHI site of the mammalianexpression vector pcDNA3.1+ (Invitrogen, Basel, Switzerland). Thespecific mutation N297Q was introduced into the CH2 domain of chCE7heavy chain using overlapping PCR and standard molecular biologytechniques. The nucleic acid and amino acid sequences for the N297Qconstruct are shown below (the mutation is underlined):

(SEQ ID NO:9)GTTTGTAAGCTTGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTAC CAAAGCACGTACCGGGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGGATCCACACAC (SEQ ID NO: 10)GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY Q STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Additionally, a further modified variant of aglycosylated N297Q variantwas made containing a Q295N mutation (i.e. containing Q295N,N297Q). Theantibodies were produced in HEK293 cells.HEK293 cells wereco-transfected with heavy and light chain containing plasmids. chCE7 wasproduced in culture dishes and purified from the harvested supernatanton a protein A sepharose column

Lysine-Based Linkers

Cadaverin-dansyl, cadaverin-biotin and cadaverin-TAMRA were purchasedfrom Zedira (Darmstadt, Germany) C2-SAc, C6-SAc, PEG-4-SAc were preparedas described in Example 1. 5-FAM cadaverin (fluorescein-5-carboxamide)was purchased from Tebu-Bio (Le Perray en Yveline, France). DBCO-amine,DBCO-PEG4-NH₂, Azide-PEG4-NH₂ and Alkyne-PEG4-NH₂ were purchased fromClick Chemistry Tools (Scottsdale, Ariz.). C2-SH and C6-SH thiol linkerswere synthesized by reduction of their corresponding disulfides asdescribed in Example 1. PEG-4-SH was synthesized by cleavage of theacetate group of PEG-4-SAc with sodium methoxide. MMAF linkers wereprepared by reacting C6-SH with maleimide-valine-citrullin-PAB-MMAF andsubsequent Boc-deprotetion. C2-DOTA and C6-DOTA linkers (thiol linkerscoupled to maleimide-DOTA) were prepared by reacting C2-SH or C6-SH withDOTA-maleimide followed by Boc deprotection. C2-fluorescein (C2-thiollilnker coupled to fluorescein maleimide) was prepared with a similarprocedure. C2-N₃ and C6-N₃ linkers were synthesized as mentioned inExample 1.

Deglycosylation of Antibodies

To antibody in PBS buffer (PBS (10×): Weight 2.1 g KH₂PO₄, 90 g NaCl,4.8 g Na₂HPO₄×2 H₂O and transfered to a 1 L glass bottle, was addedwater to a volume of 1 L. To get PBS 1×, use 100 mL PBS (10×) and addwater to a volume of 900 mL. pH was adjusted to 7.2 and filled to 1 Lwith water), and was incubated with 6 Units/mg protein of N-glycosidaseF (PNGase F) from Flavobacterium meningosepticum (Roche, Switzerland)overnight at 37° C. The enzyme was then removed bycentrifugation-dialysis (Vivaspin MWCO 50 kDa, Vivascience, Winkel,Switzerland).

Enzymatic Modification of Antibodies

1 mg/mL deglycosylated antibody in PBS was incubated with 80 equivalentsof ligand and 1 U/mL or >1 U/mL bacterial transglutaminase (BTGase,Zedira, Darmstadt, Germany) overnight at 37° C. Excess of ligand and theBTGase were removed by centrifugation-dialysis (Vivaspin MWCO 50 kDa,Vivascience, Winkel, Switzerland).

Deprotection of Protected Thiol Linkers

The method for deacetylation of the protected thiol linker is adaptedfrom published procedures (Thermo Scientific). 0.5M hydroxylamine, 25 mMEDTA is prepared in phosphate buffered saline (PBS), pH 7.2-8.5. 1 mL ofantibody-linker conjugate is combined with 100-200 μL of prepared 0.5Mhydroxylamine The mixture is incubated for 2 h at room temperature. Thereaction mixture is then be purified into PBS containing 10 mM EDTA byusing a desalting column (HiTrap Desalting column, 5 mL, GE Healthcare).

Coupling Deprotected Antibody-Linker Conjugate with MaleimideFunctionalized Moiety of Interest (Z)

Coupling of deprotected antibody-linker conjugate with maleimidefunctionalized toxin is carried out as in J. R. Junutula et al., (2008)Nat Biotechnol 26, 925. 5 equivalents per SH group of the maleimidefunctionalized ligand is combined with the deprotected antibody-linkerconjugate. The reaction is incubated at RT for 1.5 h before desaltinginto PBS.

Coupling Antibody-Linker Conjugate with Azide Group with AlkyneFunctionalized Moiety of Interest (Z)

Conjugation reactions were performed by adding amine-DBCO (50 μM, finalconcentration for 2 site-mutants; 100 μM final concentration) to theantibody-linker conjugate with azide group (20 μM) and incubating thereaction mixture for 0.5 h at room temperature. The mixture was directlyanalyzed by LC-MS.

LC NIS Analysis

LC-MS analysis was performed on a Waters LCT Premier mass spectrometer.Samples were chromatographed on an Aeris WIDEPORE XB-C18 column (3.6 μm,100 mm×2.1 mm; Phenomenex) heated to 65° C. using a linear gradient from22 to 55% A in 15 min plus 5% solvent C (solvent A: acetonitrile+0.1%formic acid, solvent B: water+0.1% formic acid, solvent C: 2-propanol)at a flow rate of 0.5 mL/min. The eluent was ionized using anelectrospray source. Data were collected with MassLynx 4.1 anddeconvolution was performed using MaxEntl. Before the LC-MS analysis, 10μg of antibody were mixed with DTT (final concentration should be 20mM). Guan-buffer (7.5M Guan-HCl, 0.1M Tris-HCl, 1 mM EDTA buffer pH 8.5(adjusted by addition of concentrated NH₄OH (28% aqueous solution) wasadded to a final volume of 50 μL. Finally, 5 μL of the mixture wereinjected.

HIC Analysis

Hydrophobic interaction chromatography (HIC) analysis was conducted onAgilent Technologies 1200 series UPLC system using a TSKgel Butyl-NPRcolumn, 4.6×35 mm, 2.5 mm particle size (Tosoh Bioscience) with a lineargradient of 100% mobile phase A (1.5 M (NH₄)₂SO₄ in 25 mM potassiumphosphate) to 70% mobile phase B (25 mM potassium phosphate, pH 7.0, 25%isopropanol) in 14 mM The flow rate was set at 1 mL/min and the columntemperature was maintained at 30° C. HIC analysis of chADC1dgl coupledto a Dansyl cadaverine substrate (see Example 3) was performed usingdouble detection by UV (280 nm) and fluorescence (λ excitation at 250nm, λ emission at 535 nm). The overall mean drug loading or DAR (DrugAntibody Ratio) is calculated as the weighted average using theintegrated areas of the constituent peaks and the drug loading of eachpeak as the weighting factor.

Western Blot Analysis

Western blot analysis: Enzymatically modified antibodies were subjectedto SDS-PAGE (12.5%) and were transferred to polyvinylidene difluoride(PVDF) membranes (Immobilon P, Millipore). After blocking with 2% bovineserum albumin (BSA) in TBST (20 mM Tris-HCl, pH 7.5, 140 mM NaCl, 0.05%Tween-20) for 2 hours at room temperature (RT), membrane was incubatedwith Streptavidin-horseradish peroxidase conjugate (High SensitivityStreptavidin-HRP diluted 1:20000; Beckman Coulter) for 30 min Membranewas washed three times with TBST for 15 min and antibodies were detectedwith Immune-Star Western C Kit chemiluminescence substrate from Biorad.

Tryptic Digest

6.67*10⁻⁹ mol protein was incubated in 100 μl 50 mM ammonium bicarbonatepH 8.0 containing 0.1% Rapidgest SF (Waters) and 0.96 μl 1M DTT at 55°C. for 30 min. After the sample was cooled to RT. 1.92 μl 1Miodoacetamide was added and the samples were incubated for 40 min at RT.The samples were then digested with 5 μg trypsin over night at 37° C.and diluted (1:1 v/v) with 1% formic acid in 10% acetonitrile andanalysed by ESI-TOF LC-MS using an ACE 3 C18, 150×3 mm column

Example 1 Synthesis of New Lysine-Based Linkers with and without SpacerGroups Materials and Methods

All solvents used for reactions were purchased as anhydrous grade fromAcros Organics (puriss., dried over molecular sieves, H₂₀<0.005%) andwere used without further purification unless otherwise stated. Solventsfor extractions, column chromatography and thin layer chromatography(TLC) were purchased as commercial grade. All non aqueous reactions wereperformed under an argon atmosphere using flame-dried glassware andstandard syringe/septa techniques. Commercially available reagents wereused without further purification. In general, reactions weremagnetically stirred and monitored by TLC performed on Merck TLC glasssheets (silica gel 60 F₂₅₄). Spots were visualized with UV light (λ=254nm) or by staining with anisaldehyde solution or KMnO₄ solution andsubsequent heating. Chromatographic purification of products wasperformed using Fluka silica gel 60 for preparative columnchromatography.

Nuclear magnetic resonance (NMR) spectra were recorded in CDCl₃, CD₃ODor D₂O either on a Bruker Av-400 or a Bruker Av-500 spectrometer at roomtemperature. The measured chemical shifts are reported in 6 (ppm) andthe residual signal of the solvent was used as the internal standard(CDCl₃ ¹H: δ=7.26 ppm, ¹³C: δ=77.0 ppm, CD₃OD ¹H: δ=3.31 ppm, ¹³C:δ=49.1 ppm, D₂O¹H: 6=4.81 ppm). All ¹³C NMR spectra were measured withcomplete proton decoupling. Data of NMR spectra are reported as follows:s=singlet, d=doublet, t=triplet, m=multiplet, dd=doublet of doublets,dt=doublet of triplets, br=broad signal. The coupling constant J isreported in Hertz (Hz). High resolution mass spectrometry (HRMS) wasperformed on a Bruker Daltonics maxis ESI-QTOF or a Varian HiResMALDIinstrument.

The analytical and preparative HPLC system used was a Merck—HitachiD-7000 system. The columns used for chromatography were either anUltimate XB-C18 (4.6×150 mm, 3 μm) or an Xbridge C18 (4.6×150 mm, 5 μm)for analytical separations operated with a flow of 1 ml/min. Forpreparative purifications, either an Ultimate XB-C18 (21.2×150 mm, 5 μm)or an Xbridge C18 (10×150 mm, 5 μm) column was used operated with a flowof 15 ml/min and 4 ml/min respectively.

Compounds 1-6 and reaction schemes are shown in FIG. 15. Compounds 7-9and reaction schemes are shown in FIG. 16A. For Compounds 10-13 andreaction schemes, see FIG. 16B.

di-tert-butyl(((2,2′-disulfanediylbis(acetyl))bis(azanediyl))bis(pentane-5,1-diyl))dicarbamate(1a)

In a solution of 2,2′-disulfanediyldiacetic acid (160 mg, 0.878 mmol),tert-butyl (5-amino-pentyl)carbamate (391 mg, 1.932 mmol) and DIPEA (920μl. 5.27 mmol) in DMF (4.9 ml), HBTU (1.33 g, 3.51 mmol) was addedportionwise at room temperature. After stirring for 5 hours, thebrownish solution was diluted with ethyl acetate (80 ml) and washed withwater (3×30 ml) and brine (lx 30 ml). The organic layer was dried undersodium sulfate, filtered and evaporated to dryness. The crude waspurified by flash column chromatography on silica using CHCl₃/EtOH 95:5to yield 420 mg (87%) of a yellow oil which solidified upon standing atroom temperature. ¹H NMR (400 MHz, CDCl₃): δ 6.91 (br, 2H), 4.68 (br,2H), 3.44 (s, 4H), 3.29 (dt, J₁=7.2 Hz, J₂=6.8 Hz, 4H), 3.10 (dt, J₁=7.7Hz, J₂=6.3 Hz, 4H), 1.64-1.31 (m, 30H). ¹³C NMR (100 MHz, CDCl₃): δ168.5, 156.1, 79.1, 42.6, 40.2, 39.8, 29.7, 28.8, 28.4, 23.9. ESI-QTOFMS m/z calculated for C₂₄H₄₆N₄O₆S₂ [M+H]⁺551.2932, measured 551.2921

di-tert-butyl(((6,6′-disulfanediylbis(hexanoyl))bis(azanediyl))bis(pentane-5,1-diyl))dicarbamate(1b)

In a solution of 6,6′-disulfanediyldihexanoic acid (250 mg, 0.849 mmol),tert-butyl (5-amino-pentyl)carbamate (412 mg, 2.038 mmol) and DIPEA(0.890 ml, 5.09 mmol) in DMF (4.7 ml), HBTU (1.29 g, 3.40 mmol) wasadded portionwise at room temperature. After stirring for 20 hours, theyellowish reaction mixture was diluted with ethyl acetate (70 ml) andwashed with cold HCl 0.1N (3×50 ml), NaHCO₃ (sat) (1×50 ml) water (1×50ml) and brine (lx 50 ml). The organic layer was dried under sodiumsulfate, filtered and evaporated to dryness. The crude was purified byflash column chromatography on silica using CHCl₃/EtOH 95:5 to yield 525mg (93%) of compound as a yellow sticky solid. ¹H NMR (400 MHz, CDCl₃):δ 5.87 (br, 2H), 4.64 (br, 2H), 3.22 (dt, J₁=7.3 Hz, J₂=6.8 Hz, 4H),3.09 (dt, J₁=8.1 Hz, J₂=6.7 Hz, 4H), 2.65 (t, J=7.2 Hz, 4H), 2.16 (t,J=7.2 Hz, 4H), 1.73-1.59 (m, 8H), 1.55-1.45 (m, 8H), 1.42 (s, 18H),1.37-1.28 (m, 4H). ¹³C NMR (100 MHz, CDCl₃): δ 172.9, 156.1, 79.0, 40.2,39.2, 38.8, 36.5, 29.7, 29.1, 28.8, 28.4, 28.0, 25.3, 23.9. ESI-QTOF MSm/z calculated for C₃₂H₆₂N₄O₆S₂ [M+1-1]⁺663.4184, measured 663.4185.

tert-butyl (5-(2-mercaptoacetamido)pentyl)carbamate (2a)

To a solution ofDi-tert-butyl(((2.2′-disulfanediylbis(acetyl))bis(azanediyl))bis(pentane-5,1-diyl))di-carbamate(390 mg, 0.478 mmol) in a mixture of tetrahydrofuran (7 ml) and water(0.74 ml), tributylphosphine (528 mg, 2.48 mmol) was added dropwise atroom temperature, within 1 min. The reaction mixture was stirred for 1 hand then the volatiles were removed under reduced pressure at 33° C. Thecrude was azeotroped once with 50 ml benzene to remove traces of waterand the residue was purified with flash column chromatography on silicawith CHCl₃/EtOH 95:5 to yield a slightly yellow clear oil. The productwas re-purified with flash column chromatography with hexane/ethylacetate 2:8 to remove oxidized tributylphosphine byproducts. Final yieldwas 180 mg (91%) of product as a colorless oil which solidified to awhite solid after storage at −25° C. ¹H NMR (400 MHz, CDCl₃): δ 6.73(br, 1H), 4.57 (br, 1H), 3.28 (dt, J₁=7.6 Hz, J₂=6.9 Hz, 2H), 3.23 (d,J=9.0 Hz, 2H), 3.11 (dt, J₁=8.1 Hz, J₂=6.6 Hz, 2H), 1.87 (t, ³J=9.0 Hz,1H), 1.61-1.47 (m, 4H), 1.43 (s, 9H), 1.40-1.30 (m, 2H). ¹³C NMR (100MHz, CDCl₃): δ 169.1, 156.1, 79.1, 40.2, 39.7, 29.7, 29.0, 28.4, 28.3,23.9. ESI-QTOF MS m/z calculated for C₁₂H₂₄N₂O₃S [M+Na]⁺299.1400,measured 299.1408.

tert-butyl (5-(6-mercaptohexanamido)pentyl)carbamate (2b)

To a solution ofdi-tert-butyl(((6,6′-disulfanediylbis(hexanoyl))bis(azanediyl))bis(pentane-5,1-diyl))di-carbamate(196 mg, 0.296 mmol) in a mixture of tetrahydrofuran (3 ml) and water(0.31 ml, 17.21 mmol), tributylphosphine (272 μl. 1.035 mmol) was addeddropwise at room temperature, within 1 min. The reaction mixture wasstirred for 1 h and then the volatiles were removed under reducedpressure at 33° C. The crude was azeotroped once with 50 ml benzene toremove traces of water and the residue was purified with flash columnchromatography on silica with chloroform/ethanol 95:5 to yield aslightly yellow clear oil. NMR revealed that the compound wascontaminated with tributylphosphine oxidized byproducts so the crude waspurified again with flash column chromatography with hexane/ethylacetate 2:8 to yield 180 mg (91%) of product as a colorless oil whichsolidified after storage at −25° C. ¹H NMR (400 MHz, CDCl₃): δ 5.88 (br,1H), 4.57 (br, 1H), 3.23 (dt, J₁=7.3 Hz, J₂=6.9 Hz, 2H), 3.09 (dt,J₁=7.8 Hz, J₂=6.5 Hz, 2H), 2.52 (dt, J₁=8.0 Hz, J₂=7.6 Hz, 2H), 2.16 (t,J=7.5 Hz, 4H), 1.69-1.57 (m, 4H), 1.56-1.46 (m, 4H), 1.43 (s, 9H),1.36-1.28 (m, 3H). ¹³C NMR (100 MHz, CDCl₃): δ 172.8, 156.1, 79.1, 40.2,39.2, 36.5, 33.6, 29.7, 29.1, 28.4, 27.9, 25.1, 24.4, 23.9. ESI-QTOF MSm/z calculated for C₁₆H₃₂N₂O₃S [M+H]⁺ 333.2206, measured 333.2198.

S-(2-((5-((tert-butoxycarbonyl)amino)pentyl)amino)-2-oxoethyl)ethanethioate(3a)

To a mixture of tert-butyl (5-(2-mercaptoacetamido)pentyl)carbamate (189mg, 0.684 mmol) and dry potassium carbonate (189 mg, 1.368 mmol) indegassed (freeze-pump-thaw) ethyl acetate (2.7 ml), acetic anhydride (77mg, 0.821 mmol) was added and the reaction was stirred for 16 h. Thereaction was then diluted with ethyl acetate (30 ml), filtered andwashed with cold water (1×15 ml) and brine (1×15 ml), dried under sodiumsulfate and evaporated to dryness. The crude was purified by flashcolumn chromatography on silica with CHC₃/EtOH 96:4 to yield 192 mg(88%) of product as a white solid. ¹H NMR (400 MHz, CDCl₃): δ 6.22 (br,1H), 4.56 (br, 1H), 3.51 (s, 2H), 3.21 (dt, J₁=7.1 Hz, J₂=6.9 Hz, 2H),3.09 (dt, J₁=7.6 Hz, J₂=6.6 Hz, 2H), 2.40 (s, 3H), 1.54-1.45 (m, 4H),1.43 (s, 9H), 1.35-1.26 (m, 2H). ¹³C NMR (100 MHz, CDCl₃): δ 190.5,168.0, 156.0, 79.1, 40.3, 39.6, 33.1, 30.3, 29.6, 29.0, 28.4, 23.8.ESI-QTOF MS m/z calculated for C₁₄H₂₆N₂O₄S [M+Na]⁺341.1505, measured341.1506.

S-(6-((5-((tert-butoxycarbonyl)amino)pentyl)amino)-6-oxohexyl)ethanethioate(3b)

To a solution of tert-butyl (5-(6-mercaptohexanamido)pentyl)carbamate(180 mg, 0.541 mmol) and dry potassium carbonate (150 mg, 1.083 mmol) indegassed (freeze-pump-thaw) ethyl acetate (2.2 ml), acetic anhydride (61μl. 0.650 mmol) was added and the reaction was stirred for 16 h. Thereaction was then diluted with ethyl acetate (20 ml), filtered andwashed with cold water (1×10 ml) and brine (1×10 ml), dried under sodiumsulfate and evaporated to dryness. The crude was purified by flashcolumn chromatography using chloroform/ethanol 96:4 to yield 182 mg(90%) of a white solid. ¹H NMR (400 MHz, CDCl₃): δ 5.68 (br, 1H), 4.61(br, 1H), 3.21 (dt, J₁=7.3 Hz, J₂=6.9 Hz, 2H), 3.09 (dt, J₁=7.7 Hz,J₂=6.4 Hz, 2H), 2.83 (t, J=7.2 Hz, 2H), 2.30 (s, 1H), 2.14 (t, J=7.2 Hz,2H), 1.67-1.44 (m, 8H), 1.42 (s, 9H), 1.40-1.27 (m, 4H). ¹³C NMR (100MHz, CDCl₃): δ 196.0, 172.8, 156.1, 79.3, 40.2, 39.2, 36.4, 30.6, 29.7,29.2, 29.1, 28.8, 28.4, 28.3, 25.1, 23.9. ESI-QTOF MS m/z calculated forC₁₈H₃₄N₂O₄S [M+H]⁺ 375.2312, measured 375.2312

S-(2-((5-aminopentyl)amino)-2-oxoethyl)ethanethioate (4a) (C2-SAclinker)

To a solution ofS-(2-((5-((tert-butoxycarbonyl)amino)pentyl)amino)-2-oxoethyl)ethanethioate(189 mg, 0.594 mmol) in dichloromethane (7.9 ml), trifluoroacetic acid(0.92 ml, 11.87 mmol) was added dropwise at 0° C. After stirring for 10min, the reaction mixture was allowed to reach room temperature where itwas stirred for 1 h. Toluene was then added (20 ml), volatiles wereremoved under reduced pressure and the residue was dried under highvacuum for 30 min to yield quantitatively a slightly yellow oil whichwas sufficiently pure when analyzed by NMR. The oil was dissolved inwater and lyophilized to give a white solid. ¹H NMR (400 MHz, CD₃OD):3.60 (s, 2H), 3.20 (t, J=6.9 Hz, 2H), 2.91 (t, J=7.6 Hz, 2H), 2.37 (s,3H), 1.72-1.61 (m, 2H), 1.59-1.50 (m, 2H), 1.45-1.35 (m, 2H). ¹³C NMR(100 MHz, CDCl₃): δ 196.3, 170.8, 40.7, 40.4, 33.9, 30.1, 29.9, 28.2,24.6. ESI-QTOF MS m/z calculated for C₉H₁₈N₂O₂S [M+1-1]⁺219.1162,measured 219.1171.

S-(6-((5-aminopentyl)amino)-6-oxohexyl)ethanethioate (4b) (C6-SAclinker)

To a solution ofS-(6-((5-((tert-butoxycarbonyl)amino)pentyl)amino)-6-oxohexyl)ethanethioate (187 mg, 0.5 mmol) in dichloromethane (6.6 ml),trifluoroacetic acid (0.77 ml, 5.34 mmol) was added dropwise at 0° C.After stirring for 10 min, the reaction mixture was allowed to reachroom temperature where it was stirred for 1 h. The volatiles wereremoved under reduced pressure at 30° C. and the residue was azeotropedwith toluene and dried under high vacuum for 30 min Lyophilizationyielded a white solid (185 mg) which was sufficiently pure by NMR. ¹HNMR (400 MHz, CD₃OD): δ 3.18 (t, J=7.0 Hz, 2H), 2.92 (t, J=7.8 Hz, 2H),2.86 (t, J=7.3 Hz, 2H), 2.30 (s, 3H), 2.17 (t, J=7.3 Hz, 2H), 1.72-1.50(m, 8H), 1.45-1.33 (m, 4H). ¹³C NMR (100 MHz, CD₃OD): 197.7, 176.2,40.7, 40.0, 37.0, 30.64, 30.61, 30.0, 29.8, 29.4, 28.3, 26.6, 24.8.ESI-QTOF MS m/z calculated for C₁₃H₂₆N₂O₂S [M+1-1]⁺275.1788, measured275.1785.

2,2′,2″-(10-(2-((2-(3-((2-((5-((tert-butoxycarbonyl)amino)pentyl)amino)-2-oxoethyl)thio)-2,5-dioxopyrrolidin-1-yl)ethyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triaceticacid (5a)

DOTA-maleimide (25 mg, 0.032 mmol) was suspended in acetonitrile (1 ml)and triethylamine was added (22.59 μl, 0.162 mmol) and after 5 min ofstirring, a clear colorless solution was formed. A solution oftert-butyl (5-(2-mercaptoacetamido)pentyl)-carbamate (10.54 mg, 0.038mmol) in 0.5 ml acetonitrile was then added and the reaction was stirredfor 1 h at which point HPLC confirmed complete consumption of startingmaterial. The solvent system used for reaction monitoring is as follows:water/0.1% TFA (solvent A), acetonitrile (solvent B); 0-5 min: 0% B,5-20 min: 0-50% B, 20-25 min: 50% B, 25-30 min 50-0% B; UV=214 nm;t_(R)=18.3 min. The reaction was then diluted with 3 ml water and waspurified by preparative HPLC with the following solvent system:water/0.1% TFA (solvent A), acetonitrile (solvent B); 0-5 min: 0% B,5-20 min: 0-50% B. The product eluted approximately at 17 min; XB-C18column; UV=214 nm. The product was obtained as a white solid afterlyophilization (19.7 mg, 77% yield). ESI-MS m/z calculated forC₃₄H₅₈N₈O₁₂S [M+H]⁺ 803.39, measured 803.40.

2,2′,2″-(10-(2-((2-)3-((6-((5-((tert-butoxycarbonyl)amino)pentyl)amino)-6-oxohexyl)thio)-2,5-dioxopyrrolidin-1-yl)ethyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triaceticacid (5b)

To a solution of DOTA-maleimide (80 mg, 0.102 mmol) and triethylamine(52.5 mg, 0.519 mmol) in acetonitrile (3.5 ml) was added a solution oftert-butyl(5-(6-mercaptohexanamido)pentyl)carbamate (40.6 mg, 0.122mmol) in acetonitrile (1.5 ml) and the reaction mixture was stirred for6 h at room temperature. Approximately half of the solvent was thenremoved under reduced pressure, water was added (3 ml) and the mixturewas purified with preparative RP HPLC with the following solvent system:water/0.1% TFA (solvent A), acetonitrile (solvent B); 0-5 min: 0% B,5-20 min: 0-50% B; t_(R)=17.4 min; UV=214 nm; XB-C18 column. The productwas obtained as a white solid after lyophilization (58 mg, 57% yield).ESI-MS m/z calculated for C₃₈H₆₆N₈O₁₂S [M+H]⁺ 859.46, measured 859.39.

5-(3-((2-((5-((tert-butoxycarbonyl)amino)pentyl)amino)-2-oxoethyl)thio)-2,5-dioxopyrrolidin-1-yl)-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoicacid (5c)

A solution of tert-butyl(5-(2-mercaptoacetamido)pentyl)carbamate (14.22mg, 0.051 mmol) in DMF (0.3 ml) was added to a solution of5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoicacid (18.32 mg, 0.043 mmol) and triethylamine (4.29 μmol) and the clearyellow solution was stirred for 3 h at room temperature. After thistime, the reaction was diluted with water (3 ml) and purified withpreparative RP HPLC with the following solvent system: water/0.1% HCOOH(solvent A), acetonitrile (solvent B); 0-5 min: 30% B, 5-20 min: 30-80%B; UV=254 nm; t_(R)=15.4 min; XB-C18 column. The product was obtained asa bright yellow solid after lyophilization (22 mg, 73% yield). ESI-MSm/z calculated for C₃₆H₃₇N₃O₁₀S [M+H]⁺ 704.23, measured 704.05.

2,2′,2″-(10-(2-((2-(3-((2-((5-aminopentyl)amino)-2-oxoethyl)thio)-2,5-dioxopyrrolidin-1-yl)ethyl)-amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triaceticacid (6a) (C2-DOTA linker)

2,2′,2″-(10-(2-((2-(3-((2-((5-((tert-butoxycarbonyl)amino)pentyl)amino)-2-oxoethyl)thio)-2,5-dioxo-pyrrolidin-1-ypethypamino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triaceticacid(18 mg, 0.022 mmol) was dissolved in a mixture of dichloromethane/TFA1:1 (2.7 ml) at 0° C. The reaction mixture was stirred for 10 min atthis temperature and was then allowed to reach room temperature where itwas stirred for 1 h at which point HPLC confirmed complete consumptionof the starting material. The volatiles were removed under reducedpressure at 20° C. and the crude was dried under high vacuum for 30 min.The residue was dissolved in 1 ml water and was purified withpreparative HPLC to provide 12.7 mg (81%) of a white solid afterlyophilization. The solvent systems that were used were the same as inthe case of 5a (t_(R)=12.8 min and t_(R)=11 6 min for analytical andpreparative HPLC respectively). ¹H NMR (500 MHz, D₂O): δ 4.26-2.89 (br,28H), 4.07 (dd, J₁=9.1 Hz, J₂=4.1 Hz, 1H), 3.58 (d, J=15.3 Hz, 1H), 3.42(d, J=15.3 Hz, 1H), 3.31 (dd, J₁=19.1 Hz, J₂=9.1 Hz, 1H), 3.22, (t,J=7.1 Hz, 2H), 2.99 (t, J=7.5 Hz, 2H), 2.74 (dd, J₁=19.1 Hz, J₂=4.1 Hz,1H), 1.72-1.64 (m, 2H), 1.60-1.52 (m, 2H), 1.44-1.36 (m, 2H). ¹³C NMR(100 MHz, D₂O): δ 178.8, 178.1, 171.3, 163.0, 162.7, 117.4, 115.1, 54.7,40.3, 39.4, 39.3, 38.3, 37.1, 35.5, 34.5, 27.7, 27.6, 26.3, 22.9 ESI-MSm/z calculated for C₂₉H₅₁N₈O₁₀S [M+H]⁺ 703.34, measured 703.32.

2,2′,2″-(10-(2-((2-(3-((6-((5-aminopentyl)amino)-6-oxohexyl)thio)-2,5-dioxopyrrolidin-1-yl)ethyl)-amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triaceticacid (6b) (C6-DOTA linker)

Compound 5b (45 mg, 0.045 mmol) was dissolved in a mixture ofdichloromethane/TFA 1:1 (5.4 ml) at 0° C. and after stirring for 10 minat this temperature, the reaction mixture was allowed to reach roomtemperature where it was stirred for 2 h. The volatiles when thenremoved under reduced pressure at 30° C. and traces of TFA were removedwith drying under high vacuum for 30 min. The residue was dissolved inwater (4 ml) and was purified with preparative RP HPLC using the methoddescribed for 5b; t_(R)=13.5 min ESI-MS m/z calculated for C₃₃H₅₈N₈O₁₀S[M+H]⁺ 759.41, measured 759.40.

5-(3-((2-((5-aminopentyl)amino)-2-oxoethyl)thio)-2,5-dioxopyrrolidin-1-yl)-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoicacid (6c) (C2-fluorescein linker)

To an ice cold suspension of 5c (10 mg, 0.014 mmol) in dichloromethane(2 ml), TFA (200 μl, 2.60 mmol) was added dropwise and the clear brightyellow solution was stirred for 10 min at 0° C. for 10 min beforeallowing it to reach room temperature where it was stirred for 40 min.Toluene was then added and the volatiles were removed under reducedpressure. The crude was purified with semi-preparative RP HPLC with thefollowing system: water/0.1% TFA (solvent A), acetonitrile (solvent B);0-3 min: 5% B, 3-10 min: 5-25% B, 10-20 min: 25% B; UV=254 nm;t_(R)=15.3 min; Xbridge column. The product was obtained as a brightyellow solid after lyophilization (6.7 mg, 78% yield). ESI-MS m/zcalculated for C₃₁H₂₉N₃O₈ S [M+H]⁺ 604.18, measured 604.04.

Synthesis of PEG Linkers

For Compounds 7-9 and reaction schemes, see FIG. 16A.

S-(2,2-dimethyl-4,12-dioxo-3,15,18,21,24-pentaoxa-5,11-diazahexacosan-26-yl)ethanethioate(7)

HBTU (421 mg, 1.11 mmol) was slowly added to a solution of2-oxo-6,9,12,15-tetraoxa-3-thiaocta-decan-18-oic acid (300 mg, 0.925mmol) and DIPEA (0.32 ml, 1.85 mmol) in DMF (4.5 ml) and the resultingsolution was stirred for 15 min. A solution of tert-butyl(5-aminopentyl)carbamate (225 mg, 1.11 mmol) in DMF (0.6 ml) was thenadded dropwise and the reaction was stirred for 14 h. The reaction wasthen diluted with 60 ml ethyl acetate and was washed with water (2×25ml) and brine (1×25 ml). The organic layer was dried under sodiumsulfate, filtered and evaporated under reduced pressure. The crude waspurified by flash column chromatography on silica usingchloroform/ethanol 95:5 to afford 380 mg (81%) of product as a slightyellow oil. ¹H NMR (400 MHz, CDCl₃): δ 6.51 (br, 1H), 4.66 (br, 1H),3.70 (t, J=5.8 Hz, 2H), 3.65-3.59 (m, 12H), 3.57 (t, J=6.6 Hz, 2H), 3.21(dt, J₁=7.3 Hz, J₂=6.9 Hz, 2H), 3.12-3.02 (m, 4H), 2.44 (t, J=5.8, 2H),2.31 (s, 3H), 1.53-1.43 (m, 4H), 1.41 (s, 9H), 1.36-1.27 (m, 2H). ¹³CNMR (100 MHz, CDCl₃): δ 195.4, 171.5, 156.0, 78.9, 70.6, 70.5, 70.3,70.2, 70.1, 69.7, 67.3, 40.3, 39.0, 36.9, 30.5, 29.6, 29.2, 28.7, 28.4,24.0. ESI-QTOF MS m/z calculated for C₂₃H₄₄N₂O₈S [M+H]⁺ 509.2891,measured 509.2884

S-(21-amino-15-oxo-3,6,9,12-tetraoxa-16-azahenicosyl)ethanethioate (8)(PEG-4-SAc linker)

To an ice cold solution ofS-(2,2-dimethyl-4,12-dioxo-3,15,18,21,24-pentaoxa-5,11-diazahexacosan-26-yl)ethanethioate(370 mg, 0.73 mmol) in dichloromethane (9.7 ml) was addedtrifluoroacetic acid (1.1 ml, 14.55 mmol). After stirring for 10 min,the reaction mixture was allowed to reach room temperature and stirredfor 2 h. The volatiles were then removed under reduced pressure,followed by drying under high vacuum. A light yellow oil resulted whichwas sufficiently pure as revealed by NMR (quantitative yield). ¹H NMR(400 MHz, CDCl₃): δ 7.79 (br, 1H), 7.23 (br, 3H), 2.33 (t, J=5.3 Hz,2H), 3.69-3.56 (m, 14H), 3.31 (dt, J₁=7.5 Hz, J₂=6.1 Hz, 2H), 3.06 (t,J=6.7 Hz, 2H), 3.03-2.92 (m, 2H), 2.58 (t, J=5.3 Hz, 2H), 2.32 (s, 3H),1.77-1.65 (m, 2H), 1.64-1.51 (m, 2H), 1.49-1.38 (m, 2H). ¹³C NMR (100MHz, CDCl₃): δ 195.7, 174.0, 70.2, 69.99, 69.97, 69.9, 69.8, 69.6, 67.2,40.0, 38.8, 35.8, 30.4, 28.1, 27.2, 26.0, 22.5. ESI-QTOF MS m/zcalculated for C₁₈H₃₆N₂O₆S [M+H]⁺ 409.2367, measured 409.2381

Tert-butyl(1-mercapto-15-oxo-3,6,9,12-tetraoxa-16-azahenicosan-21-yl)carbamate (9)

A solution of sodium methoxide 0.5 M in methanol (1.8 ml, 0.904 mmol)was added dropwise to a solution of 7 (92 mg, 0.181 mmol) in degassed(freeze-pump-thaw) methanol and the reaction was stirred at roomtemperature for 3 h. After neutralization with Amberlite 120, thesolution was filtered and evaporated to dryness. The crude was purifiedby flash column chromatography on silica using chloroform/ethanol 95:5to yield a clear colorless oil (75 mg, 89%). ¹H NMR (400 MHz, CDCl₃): δ6.48 (br, 1H), 4.64 (br, 1H), 3.71 (t, J=5.7 Hz, 2H), 3.66-3.61 (m,12H), 3.60 (t, J=6.4 Hz, 2H, partially overlapped by the previousmultiplet), 3.22 (q, J₁ J₂=7.0, 2H), 3.09 (dt, J₁=6.4 Hz, J₂=7.8 Hz,2H), 2.68 (td, J₁=6.4 Hz, J₂=8.2 Hz, 2H), 2.45 (t, J=5.7 Hz, 2H), 1.59(t, J=8.2 Hz, 1H), 1.55-1.46 (m, 4H), 1.43 (s, 9H), 1.37-1.30 (m, 2H).¹³C NMR (100 MHz, CDCl₃): δ 171.5, 156.0, 79.0, 72.8, 70.6, 70.5, 70.3,70.2, 67.3, 40.3, 39.1, 37.0, 29.6, 29.2, 28.4, 24.2, 24.0

Synthesis of Azide Linkers

For Compounds 10-13 and reaction schemes, see FIG. 16B. Compounds 11aand 11b were synthesized by following procedures already published inthe literature (Brabez N. et al, Journal of Medicinal Chemistry, 2011,54(20), 7375-7384 for 11a and Kuil J. et al, Organic and BiomolecularChemistry, 2009, 7, 4088-4094 for 11b)

tert-butyl (5-(2-azidoacetamido)pentyl)carbamate (12a)

In a solution of 2-azidoacetic acid (50 mg, 0.495 mmol), tert-butyl(5-amino-pentyl)carbamate (120 mg, 0.594 mmol) and DIPEA (128 mg, 0.989mmol) in DMF (2.7 ml), HBTU (225 mg, 0.594 mmol) was added slowly atroom temperature. After stirring for 3 hours, the slight yellow solutionwas diluted with ethyl acetate (30 ml) and was washed with HCl 0.5 M(3×15 ml) and sat. NaHCO₃ (1×15 ml) solutions, water (1×15 ml) and brine(1×15 ml). The organic layer was dried under sodium sulfate, filteredand evaporated to dryness. The crude was purified by flash columnchromatography on silica using chloroform/EtOH 95:5 to yield a clearcolorless oil (128 mg, 91%). ¹H NMR (400 MHz, CDCl₃): δ 6.35 (br, 1H),4.55 (br, 1H), 3.97 (s, 2H), 3.28 (dt, J₁=7.2 Hz, J₂=6.9 Hz, 2H), 3.11(dt, J₁=7.8 Hz, J₂=6.5 Hz, 2H), 1.61-1.47 (m, 4H), 1.43 (s, 9H),1.40-1.31 (m, 2H). ¹³C NMR (100 MHz, CDCl₃): δ 166.5, 156.0, 79.1, 52.7,40.2, 39.2, 29.7, 29.0, 28.4, 23.9.

Tert-butyl (5-(6-azidohexanamido)pentyl)carbamate (12b)

HBTU (290 mg, 0.764 mmol) was slowly added to a solution of6-azidohexanoic acid (100 mg, 0.636 mmol) and DIPEA (164 mg, 1.273 mmol)in DMF (3 ml) and the resulting solution was stirred for 15 min. Asolution of tert-butyl (5-aminopentyl)carbamate (154 mg, 0.764 mmol) inDMF (0.5 ml) was then added dropwise and the reaction was stirred for 3h. After this time, the reaction mixture was diluted with ethyl acetate(40 ml) and washed with HCl 0.5 M (3×20 ml) and sat. NaHCO₃ (1×20 ml)solutions, water (1×20 ml) and brine (lx 20 ml). The organic layer wasdried under sodium sulfate, filtered and evaporated to dryness. Thecrude was purified by flash column chromatography on silica usingchloroform/EtOH 95:5 to yield a clear colorless oil (189 mg, 87%). ¹HNMR (400 MHz, CDCl₃): δ 5.61 (br, 1H), 4.58 (br, 1H), 3.30-3.20 (m, 4H),3.10 (dt, J₁=8.0 Hz, J₂=6.8 Hz, 2H), 2.16 (t, J=7.4 Hz, 2H), 1.56-1.45(m, 4H), 1.56-1.45 (m, 4H), 1.43 (s, 9H), 1.41-1.29 (m, 4H). ¹³C NMR(100 MHz, CDCl₃): δ 172.7, 156.1, 79.1, 51.3, 40.2, 39.3, 36.5, 29.8,29.2, 28.6, 28.4, 26.4, 25.2, 23.9.

N-(5-aminopentyl)-2-azidoacetamide (13a) (C2-N₃ linker)

To an ice cold solution of 12a (19.2 mg, 0.067 mmol) in dichloromethane(0.9 ml) was added trifluoroacetic acid (153 mg, 1.346 mmol). Afterstirring for 10 min, the reaction mixture was allowed to reach roomtemperature and stirred for 2 h. Toluene (4 ml) was then added and thevolatiles were removed under reduced pressure. The crude was azeotropedagain with toluene to remove traces of TFA and was then dried under HVPfor 3 hours to yield a light yellow oil (quantitative yield) which wassufficiently pure for further use, as revealed by NMR. ¹H NMR (400 MHz,CD₃OD): δ 3.87 (s, 2H), 3.24 (t, J=7.1 Hz, 2H), 2.92 (t, J=7.5 Hz, 2H),1.72-1.63 (m, 2H), 1.62-1.53 (m, 2H), 1.46-1.36 (m, 2H). ¹³C NMR (100MHz, CD₃OD): δ 170.3, 53.1, 40.7, 40.1, 30.0, 28.3, 24.7.

N-(5-aminopentyl)-6-azidohexanamide (13b) (C6-N₃ linker)

Compound 13b was synthesized by following a similar procedure asdescribed above for 13a (starting with 22.8 mg, 0.067 mmol of 12b). ¹HNMR (400 MHz, CD₃OD): δ 3.29 (t, J=6.8 Hz, 2H), 3.19 (t, J=7 Hz, 2H),2.92 (t, J=7.7 Hz, 2H), 2.20 (t, J=7.3 Hz, 2H), 1.73-1.51 (m, 8H),1.46-1.35 (m, 4H). ¹³C NMR (100 MHz, CD₃OD): δ 176.2, 52.5, 40.7, 40.0,37.0, 30.1, 29.8, 28.3, 27.5, 26.7, 24.8.

MMAF-6Cthiol Linker Synthesis

Compounds 14-15 and reaction schemes are shown in FIG. 16C.

maleimide-valine-citrullin-PAB-MMAF+6C thiol linker (Boc protected) (14)

To a solution of maleimide-valine-citrullin-PAB-MMAF (8.8 mg, 6.61 μmol)in DMF (0.6 ml) was added 6.6 μl of a 0.1 M solution of triethylamine inDMF (0.66 μmol Et₃N), followed by the dropwise addition of a solution oftert-butyl (5-(6-mercaptohexanamido)pentyl)carbamate (3 mg, 9.02 μmol)in acetonitrile (0.3 ml). The reaction was stirred for 3 h, diluted withwater (2 ml) and purified with semi-preparative RP HPLC with thefollowing system: water/50 mM NH₄HCO₃ (solvent A), acetonitrile (solventB); 0-5 min: 40% B, 5-20 min: 40-80% B; UV=254 nm; t_(R)=10.3 min;Xbridge column. The product was obtained as a white solid afterlyophilization (8.7 mg, 79% yield).

maleimide-valine-citrullin-PAB-MMAF+6C Thiol Linker (MMAF-6C Linker)(15)

Compound 14 (8 mg, 4.81 μm) was dissolved in an ice cold solution ofdichloromethane/TFA 95:5 (8 ml). The reaction mixture was allowed toreach room temperature and stirred for 40 min after which time thevolatiles were removed under reduced pressure with the addition oftoluene. Traces of solvents were removed under high vacuum and theresidue was purified by semi-preparative HPLC with the followingsystem::water/50 mM NH₄HCO₃ (solvent A), acetonitrile (solvent B); 0-5min: 30% B, 5-20 min: 30-70% B; UV=254 nm; t_(R)=11.7 min; Xbridgecolumn. The product was obtained as a white solid after lyophilization(4.86 mg, 65% yield). ESI-QTOF MS m/z calculated for C₇₉H₁₂₇N13017S[M+2H]²⁺ 781.9670, measured 781.9667.

Example 2 BTG is Unable to Couple Linkers with Large, Hydrophobic and/orCharted Payloads in Quantitative Fashion to Antibodies 1. Coupling OfDansyl and Biotin Linkers

The structures of biotin-cadaverin and dansyl cadaverin are shown below.

Antibody chADC1 having one potential acceptor glutamine on each heavychain was degycolsylated by PNGaseF treatment and a mass of 48945 Da forunmodified, deglycosylated heavy chain was determined. The light chainremained unaffected (23341 Da found). The coupling reaction (usingstandard conditions with 1 U/mL BTG) for biotin-cadaverin and dansylcadaverin was successful however it did not go to completion

In view of the only partial coupling of biotin-cadaverin and dansylcadaverin, reaction conditions were explored in an initial step oftesting factors influencing reaction conditions. It was found that using6 U/mL BTG permitted the modification of all heavy chains ofPNGaseF-deglycosylated antibody chADC1 was achieved with either exactlyone biotin-cadaverin (MW: 328 g/mol; 328-17=311 Da; 48945+311=49256 Da,49257 Da found) or one dansyl-cadaverin (MW: 335 g/mol; 335-17=318 Da;48945+318=49263 Da, 49264 Da found) per heavy chain.

2. Coupling of Linkers with DOTA Payload is Unsuccessful

The chemical structure of a thiol linker coupled to maleimide-DOTA(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) is shownbelow (for preparation see Example 1). The molecular weight is indicatedbelow the structure.

ChADC1 antibodies and DOTA linker were reacted in the presence of BTG tomodify antibodies. Quantitative enzymatic modification of chimADC1 heavychain with short DOTA thiol linker (compound 5) by BTG could not beaccomplished (see FIG. 17A: 1 U/mL BTG, only unmodified chADC1 heavychain, 48945 Da, was found. FIG. 17B: 6 U/mL BTG, minor peak modifiedchADC1 heavy chain with one DOTA thiol linker per heavy chain, MW 702g/mol, 702-17=685 Da, 48945+685=49630 Da, 49629 Da found). Reactionconditions were explored but neither by using 1 U/mL (expected) nor byusing 6 U/mL BTG could significantly complete coupling be achieved.Prolonged incubation time could not influence the efficiency orcompletion of coupling. Compared to biotin and dansyl, DOTA has a highermolecular weight, has a more rigid structure (containing a macrocycle),and in particular is electronically negatively charged that mayinterfere with BTG activity.

3. Coupling of Linker with Fluorescein Payload is Unsuccessful

The chemical structure of lysine-based linker (cadaverin) coupled tofluorescein is shown below.

ChADC1 antibodies and cadaverin-fluorescein linker were reacted in thepresence of BTG to modify antibodies. The light chain remainedunaffected. Quantitative enzymatic modification of chADC1 heavy chainwith short fluorescein-containing linker by BTG could not beaccomplished, only unmodified chADC1 heavy chain was found. Followingexploration of reaction conditions (see Example 3), optimized conditionswere tested (80 eq ligand, 6 U/ml BTG, 1 mg/ml mAb, 18H at 37° C.) butcoupling could not be achieved. Compared to biotin and dansyl,fluorescein has a higher molecular weight, has a possibly more rigid andhydrophobic structure, notably containing a polycycle, notably atri-cycle and a further cyclic group in proximity to the site of BTGactivity.

4. Coupling of Linker with DBCO Payload is Unsuccessful

The chemical structure of the dibenzylcyclooctyne (DBCO) lysine-basedlinker (DBCO-amine) used is shown below.

ChADC1 antibodies and the DBCO lysine-based linker were reacted in thepresence of BTG to modify antibodies. The light chain remainedunaffected. Quantitative enzymatic modification of chADC1 heavy chainwith short DBCO lysine-based linker by BTG could not be accomplished,only unmodified chADC1 heavy chain was found. Following exploration ofreaction conditions (see Example 3), optimized conditions were tested(80 eq ligand, 6 U/ml BTG, 1 mg/ml mAb, 37° C.) but coupling could notbe achieved. Compared to biotin and dansyl linkers, the DBCO has apossibly more rigid structure, notably containing a polycycle, notably atri-cycle group in proximity to the site of BTG activity.

5. Coupling of Linker with TAMRA Payload is Unsuccessful

The chemical structure of a TAMRA lysine-based linker is shown below.

ChADC1 antibodies and TAMRA lysine-based linker were reacted in thepresence of BTG to modify antibodies. The light chain remainedunaffected. Quantitative enzymatic modification of chimADCl heavy chainwith short TAMRA lysine-based linker by BTG could not be accomplished,only unmodified chADC1 heavy chain was found. Following exploration ofreaction conditions (see Example 3), optimized conditions were tested(80 eq ligand, 6 U/ml BTG, 1 mg/ml mAb, 18 h at 37° C.) but at best onlypartial coupling could be achieved, with about 50% of all heavy chainshaving a linker coupled thereto. Compared to biotin and dansyl, TAMRAhas a higher molecular weight, has a possibly more rigid and hydrophobicstructure, notably containing a polycycle, notable a tri-cycle and acyclic group in proximity to the site of BTG activity.

6. Coupling of Linker with Auristatin Payload is Unsuccessful

A linker comprising the monomethyl auristatin F (MMAF), as well as avaline-citrulline dipeptide spacer, a 6-carbon spacer and a PABself-elimination spacer (MW 1562, C6-MMAF linker) were reacted in thepresence of BTG to modify chADC1 or chCE7 antibodies using optimizedreaction conditions (80 eq ligand, 6 U/ml BTG, 1 mg/ml mAb, 37° C.).Quantitative enzymatic modification of heavy chains with MMAF linker byBTG could not be accomplished. Primarily unmodified chADC1 or chCE7heavy chain was found, with a major peak corresponding to unmodifiedheavy chain (70%) and a minor peak to heavy chain with one MMAF linker(30%) for chADC1 and a major peak corresponding to unmodified heavychain (81%) and a minor peak to heavy chain with one MMAF linker (19%)for chCE7.

Example 3 Discovery of Optimized Reaction Conditions for BTG

Despite improvement with spacers, large and/or hydrophobic organicmolecules representative of cytotoxic drugs are not able to be coupledby BTG onto acceptor glutamines quantitatively (complete coupling). Toexplore the possibility that optimized reactions might permitquantitative coupling reaction parameters were explored.

All the experiments were performed on chADC1 deglycosylated with PNGaseF. Antibody concentration was to 1 mg/mL for all experiments. All theexperiments were performed using 6 U/mL of BTG. All reactions weremonitored by HIC analysis and LC-MS. Samples for HIC analysis were takenafter time periods and directly injected in HIC. Samples for MS analysiswere frozen to stop the reaction.

First, the effect of enzyme concentrations was investigated. FIG. 18Adepicts the labeling of chADC1 at different concentrations of BTGase.Higher labeling yields were achieved with increasing enzymeconcentrations for BIGase. The following exploration of reactionconditions then used optimized conditions (6 U/ml BTG, 1 mg/ml mAb, 18H)at which a plateau was reached for conjugation.

We then investigated the effect of the pH of the reaction media on theenzymatic labeling. FIGS. 18B and 18C show the labeling degrees achievedat different pH values by the BIG-mediated modification of the antibody.The most efficient labeling was detected at neutral reaction conditions(pH 7.4).

Next, the effect of temperature was investigated. FIGS. 18D and 18Edepict the labeling of chADC1 at different temperatures. Higher labelingyields were achieved at 37° C.

As a further parameter for optimization, we examined the effect of thesubstrate stoechiometry. FIGS. 18F and 18G show the labeling of chADC1with BTG employing varying amount of dansyl-cadaverin substrate.Increasing amount of the substrate resulted in a higher labeling yield.The best labeling of the antibody was achieved with dansyl-cadaverinsubstrate above 40 eq/mAb. Because of the limited solubility of thedansyl-cadaverin in aqueous buffer (containing a maximum of 10% DMSO),higher concentrations could not be investigated. Further experimentsthen used 80 equivalents of linker (molar excess based on molarity ofthe mAb), 6 U/ml BTG, 1 mg/ml mAb, 37° C. unless indicated otherwise.While equivalents (eq) are expressed as molar excess based on molarityof the mAb in the Examples herein, equivalents can also be expressed asa function of the number of acceptor glutamines in an antibody, e.g, theeq figure is divided by two for a mAb having two acceptor glutamines(e.g., one on each heavy chain) or by four for a mAb having fouracceptor glutamines (e.g., two on each heavy chain)

Example 4 Improved Lysine-Based Linkers for BTG-Mediated Direct Coupling

To explore the possibility that large, charged or hydrophobic groupsclose to the site of BTG coupling (i.e. the primary amine) influencesand inhibits BTG coupling efficiency, linkers having linearcarbon-containing frameworks acting as spacers were tested.

1. Coupling of DOTA Linkers with Spacer Group

The chemical structure of a spacer-containing thiol linker coupled tomaleimide-DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid) and a short linker were compared (for preparation see Example 1).The molecular weights are indicated below the structures.

ChADC1 antibodies and short DOTA linker (see Example 2, part 3, referredto as C2-DOTA) or DOTA linker comprising a 6-carbon spacer (referred toas C6-DOTA) were reacted in the presence of BTG to modify antibodies.Following exploration of reaction conditions (see Example 3), optimizedconditions were used (80 eq ligand, 6 U/ml BTG, 1 mg/ml mAb, 18 h at 37°C.).

Quantitative enzymatic modification of chADC1 heavy chain with C2-DOTAlinker by BTG could not be accomplished and primarily unmodified chADC1heavy chain was found, with a major peak corresponding to unmodifiedheavy chain (70%) and a minor peak to heavy chain with one C2-DOTA(30%). C6-DOTA linker comprising a 6-carbon spacer however achievedsignificantly improved coupling, with a major peak corresponding toheavy chain with one C6-DOTA (70%) and a minor peak corresponding tounmodified heavy chain (30%). Results are shown in FIG. 19A.

Example 5 PNGaseF Treatment Causes Deamidation of N297 to Generate D297

Monoclonal antibodies treated with PNGaseF are known to efficientlyremove N297-linked glycosylation. According to the literature (Suzuki etal, Glycoconjugate Journal (1995) 12:183-193) the hydrolysis of theasparaginyl amide bond by PNGase can result in the formation of anaspartic acid containing polypeptide chain. As a consequence, it ispossible that the catalytic action of PNGaseF generates a deamidatedN297 residue in the close proximity of the Q295 coupling site of BTG.

In order to investigate the extent of the deamidation reaction inducedby PNGaseF, a purified sample of PNGaseF deglycosylated chADC1enzymatically conjugated to a cadaverin-biotin linker following theprocedure of Example 3 was characterized by tryptic peptide mapanalysis. The tryptic peptides of chADC1 were generated using a standarddigestion/alkylation protocol and analyzed by nano-LC coupled toelectrospray ionization (ESI) tandem mass spectrometry. The analyseswere conducted at the PIT2 proteomic platform (Facultéde Pharmacie de laTimone, Marseille, France) on a LTQ Orbitrap Velos (Thermo Electron, SanJose, Calif.) coupled with the nanomate 3000 from Dionex. The methodchosen for the bottom-up Orbitrap analysis consisted of one full MS at30,000 of resolution in the Orbitrap cell and 10 dependant MS/MS scan inthe LTQ Velos linear trap simultaneously. The data were analyzed withProteome Discoverer (Thermo Scientific) using the Sequest software thatallows querying Database.

The full scan LC-MS analysis performed in the positive ion mode revealedthe presence of a specific N297-deamidated tryptic peptide(TKPREEQ₂₉₅YN₂₉₇STYR) including the biotin-modified glutamine atposition 295 [m/z=1983.9592 (z=1); m/z=992.4832 (z=2) and m/z=661.9912(z=3)]. The extracted ion chromatogram corresponding to the di- andtri-charged pseudo-molecular ions of the specific deamidated trypticpeptide (EIC peak at 27.70 min) is shown in FIG. 19B. The non-deamidatedpeptide [m/z=1982.9752 (z=1); m/z=991.9912 (z=2) and m/z=661.6632 (z=3)]was not detected, thus confirming that the deamidation of N297 followingPNGase F treatment was quantitative.

Full sequencing of the specific N297-deamidated tryptic peptide(TKPREEQ₂₉₅YN₂₉₇STYR) was obtained by MS/MS analysis. The MS/MS spectrumof the double-charged pseudo molecular ion is presented in FIG. 19C withthe associated fragmentation pattern (b and y ions types resulting fromfragmentation at the amide bond with charge retention on the N or Cterminus respectively).

The peptide map analysis of PNGase F deglycosylated ChADC1 conjugated toa cadaverin-biotin linker revealed a quantitative deamidation ofAsparagin 297 in the CH2 domain (the +2 position relative to acceptorglutamine Q295).

Example 6 The Environment of the Acceptor Glutamine in the Heavy ChainInfluences BTG Coupling

Despite improvement with spacers, large and/or hydrophobic organicmolecules representative of cytotoxic drugs could not be coupled by BTGonto acceptor glutamines of deglycosylated chADC1 quantitatively(complete coupling). To explore the possibility that the environment, interms of amino acids of the antibody, at the site of BTG-mediatedcoupling influences and inhibits BTG coupling efficiency, modifiedantibodies having amino acid substitutions were tested. Antibodiestreated with PNGaseF to remove N297-linked glcoyslation will have anaspartic acid at residue 297 as a result of PNGaseF-induced deamidationat the asparagine. Three antibodies having N297S substitutions weregenerated which avoided N297-linked glycosylation and in turn avoided anaspartic acid or other negatively charged residue: chADC1, SGN-35(anti-CD30) and chCE7.

Unmodified (N297), PNGaseF-deglycosylated chADC1 antibodies were reactedwith the cadaverin-fluorescein linker in the presence of BTG to modifyantibodies using optimized reaction conditions (80 eq ligand, 6 U/mlBTG, 1 mg/ml mAb, 37° C.). Quantitative enzymatic modification of chADC1heavy chain with cadaverin-fluorescein linker by BTG could not beaccomplished. Only partial modification of chADC1 heavy chains wasfound, with a substantial peak corresponding to unmodified heavy chains.However, when N297S chADC1 mutant antibodies were reacted with thecadaverin-fluorescein linker in the presence of BTG, high levels ofcoupling was observed, with a major peak corresponding to heavy chainwith one cadaverin-fluorescein linker (80%) and a minor peak tounmodified heavy chains (20%).

In another experiment, unmodified (N297), PNGaseF-deglycosylated chADC1antibodies were reacted with the cadaverin-TAMRA linker in the presenceof BTG to modify antibodies using optimized reaction conditions (80 eqligand, 6 U/ml BTG, 1 mg/ml mAb, 37° C.). Quantitative enzymaticmodification of chADC1 heavy chain with cadaverin-TAMRA linker by BTGcould not be accomplished. Partly modified chADC1 heavy chain was found,with a substantial peak corresponding to unmodified heavy chain.However, when modified N297S chADC1 antibodies were reacted with thecadaverin-TAMRA linker in the presence of BTG, quantitative coupling wasachieved, with a peak corresponding to heavy chains with onecadaverin-TAMRA linker and no uncoupled heavy chains

PNGaseF treatment modifies the side chain of the asparagine at position297 such that an aspartic acid is present at position 297 followingPNGaseF treatment. It is believed that BTG activity is inhibited bynegative electrical charges. One possible explanation is therefore thata negative electrical charge at the amino acid residue at the +2position relative to the acceptor glutamine inhibits BTG's ability tocouple onto the glutamine within the particular context of the Fc domainof the antibody. The findings therefore open the possibility to usemodified antibodies where aspartic acids are no longer present at the +2position for the coupling of large and/or hydrophobic molecules toantibodies, or more generally to modify antibodies to avoid negativeelectrical charges adjacent to the acceptor glutamine, notably at the +2position.

Example 7 Combining Spacers and Modified Antibody Constant Regions forDirect Coupling

To explore the ability of the combination of modified environment at thesubstrate (implemented by use of spacer groups in the linker) andmodified environment at the site of BTG coupling (implemented by use Fcdomain mutants) to further improve BTG coupling, linkers comprising adifferent cyclic groups with and without spacers were tested using bothunmodified or modified chimeric antibodies. The modified antibodiescontained mutations at residue N297 to avoid formation of the negativelycharged aspartic acid caused by PNGase deglysosylation. Antibodies werealso modified as Q295 in combination with N297 to form N295, Q297antibodies.

1. DOTA (Negatively Charged Payload)

ChADC1 N297S antibodies and short DOTA linker (see Example 2, part 3,referred to as C2-DOTA) or DOTA linker comprising a 6-carbon spacer(referred to as C6-DOTA) were reacted in the presence of BTG to modifyantibodies using optimized reaction conditions (80 eq ligand, 6 U/mlBTG, 1 mg/ml mAb, 37° C.). See Example 3 for optimized reactionconditions.

While enzymatic modification of chADC1 N297S heavy chain with C2-DOTA byBTG was more complete than that observed for C2-DOTA on chADC1 (PNGaseFdeglycosylated) (see Example 4), quantitative coupling could not beaccomplished and some unmodified chimADC1 heavy chain remained (see FIG.21A). However, reacting C6-DOTA linker with chADC1 N297S achieved nearquantitative coupling of all heavy chains with one C6-DOTA (see FIG.21B). The combination of improved linker and protein environmenttherefore improved the coupling observed for C6-DOTA on chADC1 (PNGaseFdeglycosylated) (see Example 4 and FIG. 21A) in which only about 70%coupling was observed.

The experiments were repeated using chCE7 Q295N,N197Q antibodies and theC6-DOTA linker using optimized reaction conditions (80 eq ligand, 6 U/mlBTG, 1 mg/ml mAb, 37° C.). The reaction achieved high levels coupling ofall heavy chains with one C6-DOTA, with a major peak corresponding toheavy chain modified with one one C6-DOTA (greater than 80%).

2. DBCO (Polycycle/Rigid Payload)

In another experiment, the chemical structure of a dibenzylcyclooctyne(DBCO) lysine-based linker comprising a “PEG” spacer and short DBCOlinkers were compared (structures shown below).

ChADC1 N297S antibodies and short DBCO linker or DBCO linker comprisinga 15-atom

PEG spacer were reacted in the presence of BTG to modify antibodiesusing optimized reaction conditions (80 eq ligand, 6 U/ml BTG, 1 mg/mlmAb, 37° C.). See Example 3 for optimized reaction conditions.

Quantitative enzymatic modification of chADC1 N297S heavy chain withshort DBCO linker by BTG could not be accomplished and primarilyunmodified chADC1 heavy chain was found, with a major peak correspondingto unmodified heavy chain (70%) and a minor peak to heavy chain with oneshort DBCO linker (30%). However, reacting DBCO linker with spacercomprising a 15-carbon PEG spacer achieved substantially quantitiative(complete) coupling of all heavy chains with one DBCO linker withspacer.

3. Cytotoxic Agent (Large, Hydrophobic Payload)

The linker tested comprised the monomethyl auristatin F (MMAF) as arepresentative large cytotoxic drug used in antibody drug conjugates, aswell as a valine-citrulline dipeptide spacer, a 6-carbon spacer and aPAB self-elimination spacer. The structure is shown below. The molecularweight is indicated below the structure.

Unmodified (N297), PNGaseF deglycosylated chADC1 and chCE7 antibodieswere reacted with the MMAF linker in the presence of BTG to modifyantibodies using optimized reaction conditions (80 eq ligand, 6 U/mlBTG, 1 mg/ml mAb, 37° C.). Quantitative enzymatic modification of chADC1heavy chain with MMAF linker by BTG could not be accomplished. Primarilyunmodified chADC1 or chCE7 heavy chain was found, with a major peakcorresponding to unmodified heavy chain (70%) and a minor peak to heavychain with one MMAF linker (30%) for chADC1 and a major peakcorresponding to unmodified heavy chain (81%) and a minor peak to heavychain with one MMAF linker (19%) for chCE7.

However, when modified N297S chADC1 antibodies were reacted with theMMAF linker in the presence of BTG achieved, quantitative coupling wasachieved, with a major peak corresponding to heavy chains with one MMAFlinker (greater than 90%). The MS spectrum of chADC1 coupled toC6-Maleimide-vc-PAB-MMAF is shown in FIG. 20A and the MS spectrum ofchADC1N297S coupled to C6-Maleimide-vc-PAB-MMAF is shown in FIG. 20B.

The experiments were repeated using chCE7 Q295N,N297Q antibodies and theMMAF linker linker using optimized reaction conditions (80 eq ligand, 6U/ml BTG, 1 mg/ml mAb, 37° C.). The reaction achieved high levelscoupling of all heavy chains with one MMAF linker, with a major peakcorresponding to heavy chain modified with one one C6-DOTA (between 86%and 91%) and a minor peak corresponding to 9%-14% unmodified heavychains.

Highly favorable reaction conditions were investigated to test whetherdirect coupling of C6-MMAF linker onto a PNGaseF-deglycosylated mAbcould be pushed to completion. Conditions tested were: mAb (1 mg/mL),160 equivalent excess of 20 mM substrate in DMSO (molar excess based onmolarity of the mAb), 6 U/mL BTGase, 200 uL reaction vol., at twoincubation durations, either T1 of 40 hours or T2 of 110 hours, in eachcase at 37° C. Amount of HC+2×C6-MMAF could be observed compared to 16 hincubation time. No difference between T1 and T2 were observed forchADC1 N297Q, and only a small difference between T1 and T2 forchCE7agl. Increasing the incubation time does not push the reaction tocompletion for PNGaseF-deglycosylated antibodies.

Example 8 Improved Processes for Direct Coupling of Auristatin

A range of processes involving different quantities of BTG and/orlinkers were tested in order to develop a process involving loweramounts of cytotoxic drug subtrate for direct coupling to antibodies.Briefly, antibody-linker conjugates were formed by quantitativeBTG-mediated coupling of the C6-MMAF linker onto chADC1 N297S andchSGN35 N297S (two glutamines per antibody) different conditions: mAb (1mg/mL), 80 eq., 40 eq., 20 eq., 10 eq. excess of 20 mM linker substratein DMSO, 4 U/mL, 2 U/mL BTG, 200 uL reaction vol., 18.5 h incubationtime at 37° C. Equivalents (eq) are indicated as molar excess based onmolarity of the mAb, thus for N297S antibodies having two acceptorglutamines, 80 eq corresponds to 40 times molar excess per acceptorglutamine.

The resulting antibodies were quantitatively functionalized withC6-MMAF, with no unfunctionalized linkers remaining, for allconcentrations of BTG when 40 eq C6-MMAF were used (i.e. 20 eq ofC6-MMAF per acceptor glutamine), while below 40 eq C6-MMAF coupllng wasno longer quantitative. Additionally, 80 equivalents of C6-MMAF yieldscomplete functionalization when 4 U/ml or 2 U/ml of BTG are used.

Example 9 Improved Linkers for a Multi-Step Process

To explore the ability of a multi-step process to improve BTG coupling,various lysine-based linker comprising a reactive group were generated.The lysine-based linker can be conjugated to an antibody via BTG,followed by reaction of the conjugated antibody with a reagentcomprising a reactive group capable of reacting with the reactive groupon the lysine-based linker. Various lysine-based linkers were designedto be capable of quantitiative coupling onto an antibody by BTG. Thelinkers lacked cyclic groups, notably polycyclic or macrocyclic groupsproximal to the primary amine (site of BTG uptake and coupling).

A first linker C2-SAc (see Example 1) comprises a lysine based moietyand a protected thiol as reactive group, having the structure asfollows.

A further linker C6-SAc (see Example 1) comprises a lysine based moiety,a protected thiol as reactive group and an additional linearcarbon-comprising framework that acts as a spacer group, and has thestructure as follows.

A further linker PEG-SAc (see Example 1) comprises a lysine basedmoiety, a protected thiol as reactive group and an additional linearcarbon-comprising PEG framework that acts as a spacer group, and has thestructure as follows.

A further linker Azide-PEG4-NH₂ comprises a lysine based moiety andspacer group together embodied as a linear carbon-comprising PEGframework, and an azide as reactive group, and has the structure asfollows.

A further linker Alkyne-PEG4-NH₂ comprises a lysine based moiety andspacer group together embodied as a linear carbon-comprising PEGframework, and an alkne as reactive group, and has the structure asfollows.

A further linker DBCO-PEG4-NH₂ comprises a lysine based moiety andspacer group together embodied as a linear carbon-comprising PEGframework, and as alkyne a dibenzylcyclooctyne (DBCO) as the reactivegroup, and has the structure as follows.

Unmodified chADC1 and chADC1 N297S antibodies and the variousreactive-group-comprising linkers were reacted in the presence of BTG tomodify antibodies using optimized reaction conditions (80 eq ligand, 6U/ml BTG, 1 mg/ml mAb, 37° C.). See Example 3 for optimized reactionconditions.

Quantitative enzymatic modification chADC1 and chADC1 N297S heavy chainswith each linker by BTG could was observed. Using 6 U/mL BTG in reactionconditions it was possible to couple the different tested thiol linkersquantitatively and stoichiometrically uniform to the heavy chain ofchADC1. The preparation for analysis is shown in the scheme below. It islikely that two peaks are appearing in the MS spectra (FIG. 21) as thebasic pH during the sample preparation for the MS measurement (see“LC-MS analysis”) can promote deacetylation of the protected thiolgroup. Partial deprotection occurred for the short thiol linker (n=1)whereas complete deprotection was observed for the long thiol linker(n=5).

Scheme (above): Deacetylation of protected thiol linkers 1 and 3 duringsample preparation for mass spectrometry. Molecular weights for bothshort (n=1) and long (n=5) protected thiol linker as well as for thecorresponding deprotected linkers 2 and 4 are indicated below thestructures.

The results in FIGS. 22A and 22B show the deconvoluted mass spectra ofchADC1 heavy chain coupled to the short (1A) and long (2B) thiol linkerFIG. 22A spectrum: Protected short linker 1: 218 g/mol, 218-17=201 Da,48945+201=49146 Da, 49145 Da found; deprotected short linker 2: 176g/mol, 176-17=159 Da, 48945+159=49104, 49103 found. FIG. 22B spectrum:Deprotected long linker 4: 232 g/mol, 232-17=215 Da, 48945+215=49160 Da,49160 Da found.

Various antibody-bound linkers were then reacted with reaction partnersto obtain final compounds. In one series of experiments, antibody-linkerconjugates were formed by quantitative BTG-mediated coupling of S-acetylprotected linker C6-SAc onto chADC1, followed by deprotection andreaction with maleimide functionalized toxin. The resulting antibodieswere successfully functionalized with toxin, accompanied by a fractionof linkers that were not functionalized.

In another series of experiments, antibody-linker conjugates were formedby quantitative BTG-mediated coupling of the Azide-PEG4-NH₂ linker ontochADC1 N297S, followed by reaction with DBCO-amine. The resultingantibodies were completely/quantitatively functionalized withDBCO-amine, with no unfunctionalized linkers remaining

Example 10 A Multiple Step Process Achieves Quantitative Coupling ontoTwo Glutamines Per Heavy Chain

We explored the ability of a multi-step process to improve BTG couplingso as to increase the number of glutamines coupled on each antibodyheavy chain. Lysine-based linkers were conjugated to antibodies modifiedto have two potential acceptor glutamines at both positions 295 and 297in a one-step or a multi-step process.

Different antibodies having N297Q substitutions were generated, chADC1N297Q, SGN-35 N297Q and chCE7 N297Q. The modified antibodies compriseone acceptor glutamine at position Q295 on each heavy chain and oneacceptor glutamine at position 297 on each heavy chain, and furthermoredo not require PNGaseF treatment to remove N297-linked glycans prior tocoupling with BTG.

Combinations of linkers C2-SAc, C6-SAc and PEG-SAc (see Example 8) andchADC1 N297Q, SGN-35 N297Q and chCE7 N297Q were reacted in the presenceof BTG to modify antibodies using optimized reaction conditions (80 eqligand, 6 U/ml BTG, 1 mg/ml mAb, 37° C.).

Quantitative (substantially complete) enzymatic modification ofunmodified chADC1 N297Q and SGN-35 N297Q heavy chains with each linkerby BTG could was observed. Each of linkers C2-SAc, C6-SAc and PEG-SAcprovided complete coupling to two glutamines on all heavy chainsLikewise, Azide-PEG4-NH₂ linker also provided complete coupling to twoglutamines on all heavy chains of chADC1 N297Q.

For comparison, MMAF linker (see Example 6) was reacted withPNGaseF-deglycosylated antibodies chADC1 N297Q in the presence of BTGusing optimized reaction conditions (80 eq ligand, 6 U/ml BTG, 1 mg/mlmAb, 37° C.). However, quantitative enzymatic modification of bothglutamines on each heavy chain with MMAF linker by BTG could not beaccomplished. Primarily chADC 1 heavy chain modified with a single MMAFlinker was found, with a major peak corresponding to modified heavychain with one MMAF linker (75%) and a minor peak to heavy chain withtwo MMAF linkers (25%) for chADC1.

Use of linkers with reactive groups capable of quantitative couplingonto two acceptor glutamines per antibody heavy chain, together withmodified antibodies having two glutamines per antibody heavy chain,provides a strategy to couple moieties of interest onto four acceptorglutamines per full antibody.

Example 11 Improved Processes for Click-Chemistry Functionalization

Equivalents of reaction partners for antibodies functionalized withAzide-PEG4-NH₂ linker were decreased in order to develop a processinvolving lower amounts of cytotoxic drug substrate. Briefly,antibody-linker conjugates were formed by quantitative BTG-mediatedcoupling of the Azide-PEG4-NH₂ linker onto chADC1 N297S (two glutaminesper antibody) and chADC1 N297Q (four glutamines per antibody), followedby reaction with DBCO-amine at room temperature at differentconcentrations: 800 μM (20 eq), 200 μM (5 eq), 100 μM (2.5 eq), 50 μM(1.25 eq), 25 μM (0.625 eq). Reaction completion was monitored atdifferent reaction times: 0.5, 1 h, 2 h, 4 h and overnight. Equivalents(eq) are indicated relative to one acceptor glutamine, thus for chADC1N297S having two acceptor glutamines, 100 μM (2.5 eq) corresponds to 5times molar excess based on molarity of the mAb. For N297Q having fourglutamines, double the amount was used (10 times molar excess based onmolarity of the mAb).

The resulting antibodies were completely/quantitatively functionalizedwith DBCO-amine, with no unfunctionalized linkers remaining for allincubation durations other than 0.5 hours when 2.5 (or more) equivalentsof DBCO-amine are used. Additionally, 5 equivalents of DBCO-amine perantibody yields complete functionalization even at 0.5 hours incubation.Furthermore, when incubated overnight, 1.25 equivalents of DBCO-amineper acceptor glumtaine achieves complete functionalization.

Example 12 BTG-Mediated Coupling of Substrates Sequence Tags on SingleChain mAbs

Recombinant proteins used included scFv (myc-tagged); affibody (dimeric,myc-tagged); nanobody (myc-tagged; non-tagged). Ligands used included:biotin-cadaverin (Zedira); desferrioxamine (Sigma Aldrich). Enzyme:MTGase (Zedira). Myc-Tag sequence: EQKLISEEDL

1. Modification of a Nanobody with Biotin-Cadaverin.

In order to assess potential acceptor glutamines, a recombinant nanobody(camelid-derived single VH domain) was incubated with MTGase andbiotin-cadaverin, and results were analysed by LC-MS. Analysis of theconjugates revealed lack of substantial labeling of the untaggednanobody (FIG. 23A). Thus, MTG does not functionalize glutamines presentwithin the backbone of the nanobody.

In contrast, LC-MS analysis revealed that the enzymatic reactionresulted in modification of the same nanobody carrying a C-terminalmyc-tag (FIG. 23B). The mass peak at 15429 has the correct mass shift of312 Da. Thus, MTG functionalizes the unique glutamine present within themyc-tag sequence. After tryptic digest, a peptide with the correct massincluding the biotin-modified glutamine could be identified (Table 6).

2. Modification of a Single Chain Variable Fragment (scFv) withBiotin-Cadaverin.

A myc-tagged scFv was incubated with MTGase and biotin-cadaverin, andresults were analysed by SDS-PAGE/western blotting. The biotinylatedscFv could be detected with streptavidin-HRP (MW 28 kDa). A degradationproduct with lower molecular weight was also detected. The modifiedpeptide could be identified after tryptic digest (Table 6).

3. Modification of a Dimeric, Myc-Tagged Affibody with Biotin-Cadaverinand Dansyl-Cadaverin.

LC-MS analysis showed quantitative modification of myc-tagged dimericaffibody with the substrates biotin-cadaverin and dansyl-cadaverin (FIG.23C). The modified biotinylated peptide was identified by massspectrometry after tryptic digest (Table 6).

TABLE 6 identified peptides of scFv, Nanobody and Affibody. Biotin- cadaverin modified Q residuesare indicated by asterisks. Protein PeptideMass (talc) Mass (found) scFv LTVLGAAAEQ*K 1410.7904 1410.7996 NanobodyTPTGQGTQVTVSSAAAEQ*K 2171.0891 2171.0613 Affibody VDANSEQ*K 1200.58081200.5671

TABLE 2 (C)_(n) X L V Y Z (CH₂)₅ NH —(C═O)—CH₂— — — Charged compound(CH₂)₅ NH —(C═O)—(CH₂)₅— — Spacer system Large, charged or hydrophobiccompound; toxin; auristatin; MMAE (CH₂)₅ NH —(C═O)—(CH₂)₅— Cleavable ornon-cleavable linker, Spacer system Large, charged or hydrophobic di- ortri-or oligo peptide; val-cit compound; toxin; auristatin; MMAE (CH₂)₅NH —(C═O)—(CH₂)₅— Cleavable or non-cleavable linker, Spacer systemLarge, charged or hydrophobic di- or tri-or oligo peptide; val-cit; orabsent compound; toxin, auristatin; MMAE (CH₂)₁₀ NH —(C═O)—(CH₂)₅—Cleavable or non-cleavable linker; Spacer system Large, charged orhydrophobic tri-or oligo peptide; val-cit; or absent compound; toxin;auristatin; MMAE (CH₂)₁₅ NH —(C═O)—(CH₂)₅— Cleavable or non-cleavablelinker; Spacer system Large, charged or hydrophobic di- or tri-or oligopeptide; val-cit; or absent compound; toxin; auristatin; MMAE (CH₂)₅ NH—CH₂—(CH₂—O—(CH₂)₄—CH₂— Cleavable or non-cleavable linker; Spacer systemLarge, charged or hydrophobic di- or tri-or oligo peptide; val-cit; orabsent compound; toxin; auristatin; MMAE (CH₂)₅ NH—CH₂—(CH₂—O—CH₂)₁₂—CH₂— Cleavable or non-cleavable linker; Spacer systemLarge, charged or hydrophobic di- or tri-or oligo peptide; val-cit; orabsent compound; toxin, auristatin; MMAE (CH₂)₅ NH—CH₂—(CH₂—O—CH₂)₁₋₂₄—CH₂— Cleavable or non-cleavable linker, Spacersystem Large, charged or hydrophobic di- or tri-or oligo peptide;val-cit; or absent compound; toxin; auristatin; MMAE (CH₂)₁₀ NH—CH₂—(CH₂—O—CH₂)₄—CH₂— Cleavable or non-cleavable linker; Spacer systemLarge, charged or hydrophobic di- or tri-or oligo peptide; val-cit; orabsent compound; toxin, auristatin; MMAE (CH₂)₁₅ NH—CH₂—(CH₂—O—(CH₂)₄—CH₂— Cleavable or non-cleavable linker; Spacer systemLarge, charged or hydrophobic di- or tri-or oligo peptide; val-cit; orabsent compound; toxin, auristatin; MMAE (CH₂)₅ NH—(C═O)—CH₂—(CH₂—O—CH₂)₄—CH₂— Cleavable or non-cleavable linker; Spacersystem Large, charged or hydrophobic di- or tri-or oligo peptide;val-cit; or absent compound; toxin; auristatin; MMAE (CH₂)₁₀ NH—(C═O)—CH₂—(CH₂—O—CH₂)₁₋₂₄—CH₂— Cleavable or non-cleavable linker;Spacer system Large, charged or hydrophobic di- or tri-or oligo peptide;val-cit; or absent compound; toxin; auristatin; MMAE (CH₂)₁₅ NH—(C═O)—CH₂—(CH₂—O—CH₂)₁₋₂₄—CH₂— Cleavable or non-cleavable linker;Spacer system Large, charged or hydrophobic di- or tri-or oligo peptide;val-cit; or absent compound; toxin; auristatin; MMAE (CH₂)₅ NH—(C═O)—(CH₂)₁₀₋₂₀— Cleavable or non-cleavable linker; Spacer systemLarge, charged or hydrophobic di- or tri-or oligo peptide; val-cit; orabsent compound; toxin; auristatin; MMAE (CH₂)₁₀ NH —(C═O)—(CH₂)₁₀₋₂₀—Cleavable or non-cleavable linker; Spacer system Large, charged orhydrophobic di- or tri-or oligo peptide; val-cit; or absent compound;toxin; auristatin; MMAE (CH₂)₁₅ NH —(C═O)—(CH₂)₁₀₋₂₀— Cleavable ornon-cleavable linker; Spacer system Large, charged or hydrophobic di- ortri-or oligo peptide; val-cit; or absent compound; toxin; auristatin;MMAE (CH₂)₅ NH —(CH₂)₁₋₆— Cleavable or non-cleavable linker, Spacersystem Large, charged or hydrophobic di- or tri-or oligo peptide;val-cit; or absent compound; toxin; auristatin; MMAE (CH₂)₁₀ NH—(CH₂)₁₋₆— Cleavable or non-cleavable linker; Spacer system Large,charged or hydrophobic di- or tri-or oligo peptide; val-cit; or absentcompound; toxin; auristatin; MMAE (CH₂)₁₅ NH —(CH₂)₁₋₆— Cleavable ornon-cleavable linker, Spacer system Large, charged or hydrophobic di- ortri-or oligo peptide; val-cit; or absent compound; toxin; auristatin;MMAE (CH₂)₅ NH —(CH₂)₁₀₋₂₀— Cleavable or non-cleavable linker; Spacersystem Large, charged or hydrophobic di- or tri-or oligo peptide;val-cit; or absent compound; toxin; auristatin; MMAE (CH₂)₁₀ NH—(CH₂)₁₀₋₂₀— Cleavable or non-cleavable linker; Spacer system Large,charged or hydrophobic di- or tri-or oligo peptide; val-cit; or absentcompound; toxin; auristatin; MMAE (CH₂)₁₅ NH —(CH₂)₁₀₋₂₀— Cleavable ornon-cleavable linker; Spacer system Large, charged or hydrophobic di- ortri-or oligo peptide; val-cit; or absent compound; toxin; auristatin;MMAE (CH₂)₅ NH —(C═O)—O—CH₂— Cleavable or non-cleavable linker, Spacersystem Large, charged or hydrophobic di- or tri-or oligo peptide;val-cit; or absent compound; toxin; auristatin; MMAE (CH₂)₁₀ NH—(C═O)—O—CH₂— Cleavable or non-cleavable linker, Spacer system Large,charged or hydrophobic di- or tri-or oligo peptide; val-cit; or absentcompound; toxin; auristatin; MMAE (CH₂)₁₅ NH —(C═O)—O—CH₂— Cleavable ornon-cleavable linker, Spacer system Large, charged or hydrophobic di- ortri-or oligo peptide; val-cit; or absent compound; toxin; auristatin;MMAE (CH₂)₅ NH —(C═O)—O—(CH₂)₂₋₂₀— Cleavable or non-cleavable linker,Spacer system Large, charged or hydrophobic di- or tri-or oligo peptide;val-cit; or absent compound; toxin; auristatin; MMAE (CH₂)₁₀ NH—(C═O)—O—(CH₂)₂₋₂₀— Cleavable or non-cleavable linker; Spacer systemLarge, charged or hydrophobic di- or tri-or oligo peptide; val-cit; orabsent compound; toxin; auristatin; MMAE (CH₂)₁₅ NH —(C═O)—O—(CH₂)₂₋₂₀—Cleavable or non-cleavable linker, Spacer system Large, charged orhydrophobic di- or tri-or oligo peptide; val-cit; or absent compound;toxin; auristatin; MMAE (CH₂)₅ NH Amino acid, di- or tri-or Cleavable ornon-cleavable linker, Spacer system Large, charged or hydrophobic oligopeptide di- or tri-or oligo peptide; val-cit; or absent compound; toxin;auristatin; MMAE (CH₂)₁₀ NH Amino acid, di- or tri-or Cleavable ornon-cleavable linker; Spacer system Large, charged or hydrophobic oligopeptide di- or tri-or oligo peptide; val-cit; or absent compound; toxin;auristatin; MMAE (CH₂)₁₅ NH Amino acid, di- or tri-or Cleavable ornon-cleavable linker; Spacer system Large, charged or hydrophobic oligopeptide di- or tri-or oligo peptide; val-cit; or absent compound; toxin;auristatin; MMAE (CH₂)₅ NH

Cleavable or non-cleavable linker, di- or tri-or oligo peptide; val-cit;or absent Spacer system; CH₂—(CH₂—O—(CH₂)₄—CH₂— Large, charged orhydrophobic compound; toxin; auristatin; MMAE (CH₂)₅ NH

Cleavable or non-cleavable linker, di- or tri-or oligo peptide; val-cit;or absent Spacer system; CH₂—(CH₂—O—(CH₂)₄—CH₂— Large, charged orhydrophobic compound; toxin; auristatin; MMAE (CH₂)₅ NH

Cleavable or non-cleavable linker; di- or tri-or oligo peptide; val-cit;or absent Spacer system; CH₂—(CH₂—O—(CH₂)₄—CH₂— Large, charged orhydrophobic compound; toxin; auristatin; MMAE —CH₂—(CH₂—O—CH₂)₃—CH₂— NH— Cleavable or non-cleavable linker; Spacer system Large, charged orhydrophobic di- or tri-or oligo peptide; val-cit; or absent compound;toxin; auristatin; MMAE —CH₂—(CH₂—O—CH₂)₃—CH₂— NH—CH₂—(CH₂—O—CH₂)₁₋₂₄—CH₂— Cleavable or non-cleavable linker; Spacersystem Large, charged or hydrophobic di- or tri-or oligo peptide;val-cit; or absent compound; toxin; auristatin; MMAE—CH₂—(CH₂—O—CH₂)₃—CH₂— NH —(C═O)—CH₂—(CH₂—O—CH₂)₁₋₂₄—CH₂— Cleavable ornon-cleavable linker, Spacer system Large, charged or hydrophobic di- ortri-or oligo peptide; val-cit; or absent compound; toxin; auristatin;MMAE —O—(CH₂)₁₋₅ NH — Cleavable or non-cleavable linker, Spacer systemLarge, charged or hydrophobic di- or tri-or oligo peptide; val-cit; orabsent compound; toxin; auristatin; MMAE —O—CH₂—(CH₂—O—CH₂)₃—CH₂— NH —Cleavable or non-cleavable linker; Spacer system Large, charged orhydrophobic di- or tri-or oligo peptide; val-cit; or absent compound;toxin; auristatin; MMAE —O—CH₂—(CH₂—O—CH₂)₃—CH₂— NH—CH₂—(CH₂—O—CH₂)₁₋₂₄—CH₂— Cleavable or non-cleavable linker, Spacersystem Large, charged or hydrophobic di- or tri-or oligo peptide;val-cit; or absent compound; toxin; auristatin; MMAE—O—CH₂—(CH₂—O—CH₂)₃—CH₂— NH —(C═O)—CH₂—(CH₂—O—CH₂)₁₋₂₄—CH₂— Cleavable ornon-cleavable linker, Spacer system Large, charged or hydrophobic di- ortri-or oligo peptide; val-cit; or absent compound; toxin; auristatin;MMAE (CH₂)₄—CH(NH₂)—(C═O)— NH — — Charged compound (CH₂)₄—CH(NH₂)—(C═O)—NH — di- or tri-or oligo peptide; val-cit Spacer system Large, chargedor hydrophobic compound; toxin; auristatin, MMAE (CH₂)₄—CH(NH₂)—(C═O)—NH —(CH₂)₁₋₆— Cleavable or non-cleavable linker; Spacer system Large,charged or hydrophobic di- or tri-or oligo peptide; val-cit; or absentcompound; toxin; auristatin; MMAE (CH₂)₄—CH(NH₂)—(C═O)— NH —(CH₂)₅—Cleavable or non-cleavable linker; Spacer system Large, charged orhydrophobic; di- or tri-or oligo peptide; val-cit; or absent compound;toxin; auristatin; MMAE (CH₂)₄—CH(NH₂)—(C═O)— NH—CH₂—(CH₂—O—CH₂)₁₋₂₄—CH₂— Cleavable or non-cleavable linker; Spacersystem Large, charged or hydrophobic di- or tri-or oligo peptide;val-cit; or absent compound; toxin; auristatin; MMAE (CH₂)₅ NH — —Spacer system; Large, charged or hydrophobic (CH₂)₅ compound; toxin;auristatin; MMAF (CH₂)₆₋₁₀ NH — — Spacer system; Large, charged orhydrophobic (CH₂)₅ compound; toxin; auristatin, MMAF (CH₂)₁₅ NH — —Spacer system; Large, charged or hydrophobic (CH₂)₅ compound; toxin;auristatin; MMAF —CH₂—(CH₂—O—CH₂)₃—CH₂— NH — — Spacer system; Large,charged or hydrophobic (CH₂)₅ compound; toxin; auristatin; MMAF (CH₂)₅NH —(C═O)—CH₂— — Spacer system; Large, charged or hydrophobic (CH₂)₅compound; toxin, auristatin; MMAF (CH₂)₅ NH —(C═O)—(CH₂)₅— — Spacersystem; Large, charged or hydrophobic (CH₂)₅ compound; toxin;auristatin; MMAF (CH₂)₅ NH —CH₂—(CH₂—O—CH₂)₁₋₂₄—CH₂— — Spacer system;Large, charged or hydrophobic (CH₂)₅ compound; toxin; auristatin; MMAF—O—(CH₂)₁₋₅ NH —CH₂—(CH₂O—CH₂)₁₋₂₄—CH₂— — Spacer system; Large, chargedor hydrophobic (CH₂)₅ compound; toxin; auristatin; MMAF—O—CH₂—(CH₂—O—CH₂)₃—CH₂— NH —CH₂—(CH₂—O—CH₂)₁₋₂₄—CH₂— — Spacer system;Large, charged or hydrophobic (CH₂)₅ compound; toxin; auristatin; MMAF—O—CH₂—(CH₂—O—CH₂)₃—CH₂— NH —(C═O)—CH₂— — Spacer system; Large, chargedor hydrophobic (CH₂)₅ compound; toxin; auristatin; MMAF—O—CH₂—(CH₂—O—CH₂)₃—CH₂— NH —(C═O)—(CH₂)₅— — Spacer system; Large,charged or hydrophobic (CH₂)₅ compound; toxin; auristatin, MMAF—CH₂—(CH₂—O—CH₂)₃—CH₂— NH —CH₂—(CH₂—O—CH₂)₁₋₂₄—CH₂— — Spacer system;Large, charged or hydrophobic (CH₂)₅ compound; toxin; auristatin; MMAF(CH₂)₅ NH — Non-cleavable linker; (CH₂)₅ — Large, charged or hydrophobiccompound; toxin; auristatin; MMAF (CH₂)₆₋₁₀ NH — Non-cleavable linker;(CH₂)₅ — Large, charged or hydrophobic compound; toxin; auristatin; MMAF(CH₂)₁₅ NH — Non-cleavable linker; (CH₂)₅ — Large, charged orhydrophobic compound; toxin; auristatin; MMAF —CH₂—(CH₂—O—CH₂)₃—CH₂— NH— Non-cleavable linker; (CH₂)₅ — Large, charged or hydrophobic compound;toxin; auristatin; MMAF (CH₂)₅ NH —(C═O)—CH₂— Non-cleavable linker;(CH₂)₅ — Large, charged or hydrophobic compound; toxin; auristatin; MMAF(CH₂)₅ NH —(C═O)—(CH₂)₅— Non-cleavable linker; (CH₂)₅ — Large, chargedor hydrophobic compound; toxin; auristatin; MMAF (CH₂)₅ NH—(C═O)—(CH₂)₅— Non-cleavable linker; (CH₂)₅ — Large, charged orhydrophobic compound; toxin; auristatin; MMAF (CH₂)₅ NH —(C═O)—(CH₂)₅—Non-cleavable linker; (CH₂)₅ — Large, charged or hydrophobic compound;toxin; auristatin; MMAF (CH₂)₅ NH —CH₂—(CH₂—O—CH₂)₁₋₂₄—CH₂—Non-cleavable linker; (CH₂)₅ — Large, charged or hydrophobic compound;toxin; auristatin; MMAF —O—(CH₂)₁₋₅ NH —CH₂—(CH₂—O—CH₂)₁₋₂₄—CH₂—Non-cleavable linker; (CH₂)₅ — Large, charged or hydrophobic compound;toxin; auristatin, MMAF —O—CH₂—(CH₂—O—CH₂)₃—CH₂— NH—CH₂—(CH₂—O—CH₂)₁₋₂₄—CH₂— Non-cleavable linker; (CH₂)₅ — Large, chargedor hydrophobic compound; toxin; auristatin; MMAF—O—CH₂—(CH₂—O—CH₂)₃—CH₂— NH —(C═O)—CH₂— Non-cleavable linker; (CH₂)₅ —Large, charged or hydrophobic compound; toxin; auristatin; MMAF—O—CH₂—(CH₂—O—CH₂)₃—CH₂— NH —(C═O)—(CH₂)₅— Non-cleavable linker; (CH₂)₅— Large, charged or hydrophobic compound; toxin; auristatin; MMAF—CH₂—(CH₂—O—CH₂)₃—CH₂— NH —CH₂—(CH₂—O—CH₂)₁₋₂₄—CH₂— Non-cleavablelinker; (CH₂)₅ — Large, charged or hydrophobic compound; toxin;auristatin; MMAF (CH₂)₅ O —(C═O)—CH₂— — — Charged compound (CH₂)₅ O—(C═O)—(CH₂)₅— — Spacer system Large, charged or hydrophobic compound;toxin; auristatin; MMAF (CH₂)₅ O —(C═O)—(CH₂)₅— Cleavable ornon-cleavable linker; Spacer system Large, charged or hydrophobic di- ortri-or oligo peptide; val-cit compound; toxin; auristatin; MMAE (CH₂)₅ O—(C═O)—(CH₂)₅— Cleavable or non-cleavable linker; Spacer system Large,charged or hydrophobic di- or tri-or oligo peptide; val-cit; or absentcompound; toxin; auristatin; MMAE (CH₂)₁₀ O —(C═O)—(CH₂)₅— Cleavable ornon-cleavable linker; Spacer system Large, charged or hydrophobic di- ortri-or oligo peptide; val-cit; or absent compound; toxin; auristatin;MMAE (CH₂)₁₅ O —(C═O)—(CH₂)₅— Cleavable or non-cleavable linke; Spacersystem Large, charged or hydrophobic di- or tri-or oligo peptide;val-cit; or absent compound; toxin; auristatin; MMAE (CH₂)₅ O—CH₂—(CH₂—O—CH₂)₄—CH₂— Cleavable or non-cleavable linker; Spacer systemLarge, charged or hydrophobic di- or tri-or oligo peptide; val-cit; orabsent compound; toxin; auristatin; MMAE (CH₂)₅ O—CH₂—(CH₂—O—CH₂)₁₂—CH₂— Cleavable or non-cleavable linker; Spacer systemLarge, charged or hydrophobic di- or tri-or oligo peptide; val-cit; orabsent compound; toxin; auristatin; MMAE (CH₂)₅ O—CH₂—(CH₂—O—CH₂)₁₋₂₄—CH₂— Cleavable or non-cleavable linker; Spacersystem Large, charged or hydrophobic di- or tri-or oligo peptide;val-cit; or absent compound; toxin; auristatin; MMAE (CH₂)₁₀ O—CH₂—(CH₂—O—CH₂)₄—CH₂— Cleavable or non-cleavable linker; Spacer systemLarge, charged or hydrophobic di- or tri-or oligo peptide; val-cit; orabsent compound; toxin; auristatin; MMAE (CH₂)₁₅ O—CH₂—(CH₂—O—CH₂)₄—CH₂— Cleavable or non-cleavable linker; Spacer systemLarge, charged or hydrophobic di- or tri-or oligo peptide; val-cit; orabsent compound; toxin; auristatin; MMAE (CH₂)₅ O—(C═O)—CH₂—(CH₂—O—CH₂)₄—CH₂— Cleavable or non-cleavable linker; Spacersystem Large, charged or hydrophobic di- or tri-or oligo peptide;val-cit; or absent compound; toxin; auristatin; MMAE (CH₂)₁₀ O—(C═O)—CH₂—(CH₂—O—CH₂)₁₋₂₄—CH₂— Cleavable or non-cleavable linker;Spacer system Large, charged or hydrophobic di- or tri-or oligo peptide;val-cit; or absent compound; toxin; auristatin; MMAE (CH₂)₁₅ O—(C═O)—CH₂—(CH₂—O—CH₂)₁₋₂₄—CH₂— Cleavable or non-cleavable linker;Spacer system Large, charged or hydrophobic di- or tri-or oligo peptide;val-cit; or absent compound; toxin; auristatin; MMAE (CH₂)₅ O—(C═O)—(CH₂)₁₀₋₂₀— Cleavable or non-cleavable linker; Spacer systemLarge, charged or hydrophobic di- or tri-or oligo peptide; val-cit; orabsent compound; toxin; auristatin; MMAE (CH₂)₁₀ O —(C═O)—(CH₂)₁₀₋₂₀—Cleavable or non-cleavable linker; Spacer system Large, charged orhydrophobic di- or tri-or oligo peptide; val-cit; or absent compound;toxin; auristatin; MMAE (CH₂)₁₅ O —(C═O)—(CH₂)₁₀₋₂₀— Cleavable ornon-cleavable linker; Spacer system Large, charged or hydrophobic di- ortri-or oligo peptide; val-cit; or absent compound; toxin; auristatin;MMAE (CH₂)₅ O —(CH₂)₁₋₆— Cleavable or non-cleavable linker; Spacersystem Large, charged or hydrophobic di- or tri-or oligo peptdie;val-cit; or absent compound; toxin; auristatin; MMAE (CH₂)₁₀ O—(CH₂)₁₋₆— Cleavable or non-cleavable linker; Spacer system Large,charged or hydrophobic di- or tri-or oligo peptdie; val-cit; or absentcompound; toxin; auristatin; MMAE (CH₂)₁₅ O —(CH₂)₁₋₆— Cleavable ornon-cleavable linker; Spacer system Large, charged or hydrophobic di- ortri-or oligo peptide; val-cit; or absent compound; toxin; auristatin;MMAE (CH₂)₅ O —(CH₂)₁₀₋₂₀— Cleavable or non-cleavable linker; Spacersystem Large, charged or hydrophobic di- or tri-or oligo peptide;val-cit; or absent compound; toxin; auristatin; MMAE (CH₂)₁₀ O—(CH₂)₁₀₋₂₀— Cleavable or non-cleavable linker; Spacer system Large,charged or hydrophobic di- or tri-or oligo peptide; val-cit; or absentcompound; toxin; auristatin; MMAE (CH₂)₁₅ O —(CH₂)₁₀₋₂₀— Cleavable ornon-cleavable linker, Spacer system Large, charged or hydrophobic di- ortri-or oligo peptide; val-cit; or absent compound; toxin; auristatin;MMAE (CH₂)₅ O —(C═O)—O—CH₂— Cleavable or non-cleavable linker; Spacersystem Large, charged or hydrophobic di- or tri-or oligo peptide;val-cit; or absent compound; toxin; auristatin; MMAE (CH₂)₁₀ O—(C═O)—O—CH₂— Cleavable or non-cleavable linker; Spacer system Large,charged or hydrophobic di- or tri-or oligo peptide; val-cit; or absentcompound; toxin; auristatin; MMAE (CH₂)₁₅ O —(C═O)—O—CH₂— Cleavable ornon-cleavable linker; Spacer system Large, charged or hydrophobic di- ortri-or oligo peptide; val-cit; or absent compound; toxin; auristatin;MMAE (CH₂)₅ O —(C═O)—O—(CH₂)₂₋₂₀— Cleavable or non-cleavable linker;Spacer system Large, charged or hydrophobic di- or tri-or oligo peptide;val-cit; or absent compound; toxin; auristatin; MMAE (CH₂)₁₀ O—(C═O)—O—(CH₂)₂₋₂₀— Cleavable or non-cleavable linker; Spacer systemLarge; charged or hydrophobic di- or tri-or oligo peptide; val-cit; orabsent compound; toxin; auristatin; MMAE (CH₂)₁₅ O —(C═O)—O—(CH₂)₂₋₂₀—Cleavable or non-cleavable linker; Spacer system Large, charged orhydrophobic di- or tri-or oligo peptide; val-cit; or absent compound;toxin; auristatin; MMAE (CH₂)₅ O Amino acid, di- or tri-or Cleavable ornon-cleavable linker; Spacer system Large, charged or hydrophobic oligopeptide di- or tri-or oligo peptide; val-cit; or absent compound; toxin;auristatin; MMAE (CH₂)₁₀ O Amino acid, di- or tri-or Cleavable ornon-cleavable linker; Spacer system Large, charged or hydrophobic oligopeptide di- or tri-or oligo peptide; val-cit; or absent compound; toxin;auristatin; MMAE (CH₂)₁₅ O Amino acid, di- or tri-or Cleavable ornon-cleavable linker; Spacer system Large, charged or hydrophobic oligopeptide di- or tri-or oligo peptide; val-cit; or absent compound; toxin;auristatin; MMAE (CH₂)₅ O

Cleavable or non-cleavable linker; di- or tri-or oligo peptide; val-cit;or absent Spacer system; CH₂—(CH₂—O—(CH₂)₄—CH₂— Large, charged orhydrophobic compound; toxin; auristatin; MMAE (CH₂)₅ O

Cleavable or non-cleavable linker; di- or tri-or oligo peptide; val-cit;or absent Spacer system; CH₂—(CH₂—O—CH₂)₄—CH₂— Large, charged orhydrophobic compound; toxin; auristatin; MMAE (CH₂)₅ O

Cleavable or non-cleavable linker; di- or tri-or oligo peptide; val-cit;or absent Spacer system; CH₂—(CH₂—O—CH₂)₄—CH₂— Large, charged orhydrophobic compound; toxin; auristatin; MMAE —CH₂—(CH₂—O—CH₂)₃—CH₂— O —Cleavable or non-cleavable linker; Spacer system Large, charged orhydrophobic di- or tri-or oligo peptide; val-cit; or absent compound;toxin; auristatin; MMAE —CH₂—(CH₂—O—CH₂)₃—CH₂— O—CH₂—(CH₂—O—CH₂)₁₋₂₄—CH₂— Cleavable or non-cleavable linker; Spacersystem Large, charged or hydrophobic di- or tri-or oligo peptide;val-cit; or absent compound; toxin; auristatin; MMAE—CH₂—(CH₂—O—CH₂)₃—CH₂— O —(C═O)—CH₂—(CH₂—O—CH₂)₁₋₂₄—CH₂— Cleavable ornon-cleavable linker; Spacer system Large, charged or hydrophobic di- ortri-or oligo peptide; val-cit; or absent compound; toxin; auristatin; EMMA —O—(CH₂)₁₋₅ O — Cleavable or non-cleavable linker; Spacer systemLarge, charged or hydrophobic di- or tri-or oligo peptide; val-cit; orabsent compound; toxin; auristatin; MMAE —O—CH₂—(CH₂—O—CH₂)₃—CH₂— O —Cleavable or non-cleavable linker; Spacer system Large, charged orhydrophobic di- or tri-or oligo peptide; val-cit; or absent compound;toxin; auristatin; MMAE —O—CH₂—(CH₂—O—CH₂)₃—CH₂— O—CH₂—(CH₂—O—CH₂)₁₋₂₄—CH₂— Cleavable or non-clevable linker; Spacersystem Large, charged or hydrophobic di- or tri-or oligo peptide;val-cit; or absent compound; toxin; auristatin; MMAE—O—CH₂—(CH₂—O—CH₂)₃—CH₂— O —(C═O)—CH₂—(CH₂—O—CH₂)₁₋₂₄—CH₂— Cleavable ornon-cleavable linker; Spacer system Large, charged or hydrophobic di- ortri-or oligo peptide; val-cit; or absent compound; toxin; auristatin;MMAE (CH₂)₄—CH(NH₂)—(C═O)— O — — Charged compound (CH₂)₄—CH(NH₂)—(C═O)—O — di- or tri-or oligo peptide; val-cit Spacer system Large, charged orhydrophobic compound; toxin; auristatin; MMAE (CH₂)₄—CH(NH₂)—(C═O)— O—(CH₂)₁₋₆— Cleavable or non-cleavable linker; Spacer system Large,charged or hydrophobic di- or tri-or oligo peptide; val-cit; or absentcompound; toxin; auristatin; MMAE (CH₂)₄—CH(NH₂)—(C═O)— O —(CH₂)₅—Cleavable or non-cleavable linker; Spacer system Large, charged orhydrophobic di- or tri-or oligo peptide; val-cit; or absent compound;toxin; auristatin; MMAE (CH₂)₄—CH(NH₂)—(C═O)— O —CH₂—(CH₂O—CH₂)₁₋₂₄—CH₂—Cleavable or non-cleavable linker; Spacer system Large, charged orhydrophobic di- or tri-or oligo peptide; val-cit; or absent compound;toxin; auristatin; MMAF (CH₂)₅ O — — Spacer system; Large, charged orhydrophobic (CH₂)₅ compound; toxin; auristatin; MMAF (CH₂)₆₋₁₀ O — —Spacer system; Large, charged or hydrophobic (CH₂)₅ compound; toxin;auristatin; MMAF (CH₂)₁₅ O — — Spacer system; Large, charged orhydrophobic (CH₂)₅ compound; toxin; auristatin; MMAF—CH₂—(CH₂—O—CH₂)₃—CH₂— O — — Spacer system; Large, charged orhydrophobic (CH₂)₅ compound; toxin; auristatin; MMAF (CH₂)₅ O—(C═O)—CH₂— — Spacer system; Large, charged or hydrophobic (CH₂)₅compound; toxin; auristatin; MMAF (CH₂)₅ O —(C═O)—(CH₂)₅— — Spacersystem; Large, charged or hydrophobic (CH₂)₅ compound; toxin;auristatin; MMAF (CH₂)₅ O —CH₂—(CH₂—O—CH₂)₁₋₂₄—CH₂— — Spacer system;Large, charged or hydrophobic (CH₂)₅ compound; toxin; auristatin; MMAF—O—(CH₂)₁₋₅ O —CH₂—(CH₂—O—CH₂)₁₋₂₄—CH₂— — Spacer system; Large, chargedor hydrophobic (CH₂)₅ compound; toxin; auristatin; MMAF—O—CH₂—(CH₂—O—CH₂)₃—CH₂— O —CH₂—(CH₂—O—CH₂)₁₋₂₄—CH₂— — Spacer system;Large, charged or hydrophobic (CH₂)₅ compound; toxin; auristatin; MMAF—O—CH₂—(CH₂—O—CH₂)₃—CH₂— O —(C═O)—CH₂— — Spacer system; Large, chargedor hydrophobic (CH₂)₅ compound; toxin; auristatin; MMAF—O—CH₂—(CH₂—O—CH₂)₃—CH₂— O —(C═O)—(CH₂)₅— — Spacer system; Large,charged or hydrophobic (CH₂)₅ compound; toxin; auristatin; MMAF—CH₂—(CH₂—O—CH₂)₃—CH₂— O —CH₂—(CH₂—O—CH₂)₁₋₂₄—CH₂— — Spacer system;Large, charged or hydrophobic (CH₂)₅ compound; toxin; auristatin; MMAF(CH₂)₅ O — Non-cleavable linker; (CH₂)₅ — Large, charged or hydrophobiccompound; toxin; auristatin; MMAF (CH₂)₆₋₁₀ O — Non-cleavable linker;(CH₂)₅ — Large, charged or hydrophobic compound; toxin; auristatin; MMAF(CH₂)₁₅ O — Non-cleavable linker; (CH₂)₅ — Large, charged or hydrophobiccompound; toxin; auristatin; MMAF —CH₂—(CH₂—O—CH₂)₃—CH₂— O —Non-cleavable linker; (CH₂)₅ — Large, charged or hydrophobic compound;toxin; auristatin; MMAF (CH₂)₅ O —(C═O)—CH₂— Non-cleavable linker;(CH₂)₅ — Large, charged or hydrophobic compound; toxin; auristatin; MMAF(CH₂)₅ O —(C═O)—(CH₂)₅— Non-cleavable linker; (CH₂)₅ — Large, charged orhydrophobic compound; toxin; auristatin; MMAF (CH₂)₅ O —(C═O)—(CH₂)₅—Non-cleavable linker; (CH₂)₅ — Large, charged or hydrophobic compound;toxin; auristatin; MMAF (CH₂)₅ O —(C═O)—(CH₂)₅— Non-cleavable linker;(CH₂)₅ — Large, charged or hydrophobic compound; toxin; auristatin; MMAF(CH₂)₅ O —CH₂—(CH₂—O—CH₂)₁₋₂₄—CH₂— Non-cleavable linker; (CH₂)₅ — Large,charged or hydrophobic compound; toxin; auristatin; MMAF —O—(CH₂)₁₋₅ O—CH₂—(CH₂—O—CH₂)₁₋₂₄—CH₂— Non-cleavable linker; (CH₂)₅ — Large, chargedor hydrophobic compound; toxin; auristatin; MMAF—O—CH₂—(CH₂—O—CH₂)₃—CH₂— O —CH₂—(CH₂—O—CH₂)₁₋₂₄—CH₂— Non-cleavablelinker; (CH₂)₅ — Large, charged or hydrophobic compound; toxin;auristatin; MMA —O—CH₂—(CH₂—O—CH₂)₃—CH₂— O —(C═O)—CH₂— Non-cleavablelinker; (CH₂)₅ — Large, charged or hydrophobic compound; toxin;auristatin; MMAF —O—CH₂—(CH₂—O—CH₂)₃—CH₂— O —(C═O)—(CH₂)₅— Non-cleavablelinker; (CH₂)₅ — Large, charged or hydrophobic compound; toxin;auristatin; MMAF —CH₂—(CH₂—O—CH₂)₃—CH₂— O —CH₂(CH₂—O—CH₂)₁₋₂₄—CH₂—Non-cleavable linker; (CH₂)₅ — Large, charged or hydrophobic compound;toxin; auristatin; MMAF (CH₂)₅ S —(C═O)—CH₂— — — Charged compound (CH₂)₅S —(C═O)—(CH₂)₅— — Spacer system Large, charged or hydrophobic compound;toxin; auristatin; MMAE (CH₂)₅ S —(C═O)—(CH₂)₅— Cleavable ornon-cleavable linker; Spacer system Large, charged or hydrophobic di- ortri-or oligo peptide; val-cit compound; toxin; auristatin; MMAE (CH₂)₅ S—(C═O)—(CH₂)₅— Cleavable or non-cleavable linker; Spacer system Large,charged or hydrophobic di- or tri-or oligo peptide; val-cit; or absentcompound; toxin; auristatic; MMAE (CH₂)₁₀ S —(C═O)—(CH₂)₅— Cleavable ornon-cleavable linker; Spacer system Large, charged or hydrophobic di- ortri-or oligo peptide; val-cit; or absent compound; toxin; auristatin;MMAE (CH₂)₁₅ S —(C═O)—(CH₂)₅— Cleavable or non-cleavable linker; Spacersystem Large, charged or hydrophobic di- or tri-or oligo peptide;val-cit; or absent compound; toxin; auristatin; MMAE (CH₂)₅ S—CH₂—(CH₂—O—CH₂)₄—CH₂— Cleavable or non-cleavable linker; Spacer systemLarge, charged or hydrophobic di- or tri-or oligo peptide; val-cit; orabsent compound; toxin; auristatin; MMAE (CH₂)₅ S—CH₂—(CH₂—O—CH₂)₁₂—CH₂— Cleavable or non-cleavable linker; Spacer systemLarge, charged or hydrophobic di- or tri-or oligo peptide; val-cit; orabsent compound; toxin; auristatin; MMAE (CH₂)₅ S—CH₂—(CH₂—O—CH₂)₁₋₂₄—CH₂— Cleavable or non-cleavable linker; Spacersystem Large, charged or hydrophobic di- or tri-or oligo peptide;val-cit; or absent compound; toxin; auristatin; MMAE (CH₂)₁₀ S—CH₂—(CH₂—O—CH₂)₄—CH₂— Cleavable or non-cleavable linker; Spacer orsystem Large, charged or hydrophobic di- or tri-or oligo peptide;val-cit; or absent compound; toxin; auristatin; MMAE (CH₂)₁₅ S—CH₂—(CH₂—O—CH₂)₄—CH₂— Cleavable or non-cleavable linker; Spacer systemLarge, charged or hydrophobic di- or tri-or oligo peptide; val-cit; orabsent compound; toxin; auristatin; MMAE (CH₂)₅ S—(C═O)—CH₂—(CH₂—O—CH₂)₄—CH₂— Cleavable or non-cleavable linker; Spacersystem Large, charged or hydrophobic di- or tri-or oligo peptide;val-cit; or absent compound; toxin; auristatin; MMAE (CH₂)₁₀ S—(C═O)—CH₂—(CH₂—O—CH₂)₁₋₂₄—CH₂— Cleavable or non-cleavable linker;Spacer system Large, charrged or hydrophobic di- or tri-or oligopeptide; val-cit; or absent compound; toxin; auristatin; MMAE (CH₂)₁₅ S—(C═O)—CH₂—(CH₂—O—CH₂)₁₋₂₄—CH₂— Cleavable or non-cleavable linker;Spacer system Large, charged or hydrophobic di- or tri-or oligo peptide;val-cit; or absent compound; toxin; auristatin; MMAE (CH₂)₅ S—(C═O)—(CH₂)₁₀₋₂₀— Cleavable or non-cleavable linker; Spacer systemLarge, charged or hydrophobic di- or tri-or oligo peptide; val-cit; orabsent compound; toxin; auristatin; MMAE (CH₂)₁₀ S —(C═O)—(CH₂)₁₀₋₂₀—Cleavable or non-cleavable linker; Spacer system Large, charged orhydrophobic di- or tri-or oligo peptide; val-cit; or absent compound;toxin; auristatin; MMAE (CH₂)₁₅ S —(C═O)—(CH₂)₁₀₋₂₀— Cleavable ornon-cleavable linker; Spacer system Large, charged or hydrophobic di- ortri-or oligo peptide; val-cit; or absent compound; toxin; auristatin;MMAE (CH₂)₅ S —(CH₂)₁₋₆— Cleavable or non-cleavable linker; Spacersystem Large, charged or hydrophobic di- or tri-or oligo peptide;val-cit; or absent compound; toxin; auristatin; MMAE (CH₂)₁₀ S—(CH₂)₁₋₆— Cleavable or non-cleavable linker; Spacer system Large,charged or hydrophobic di- or tri-or oligo peptide; val-cit; or absentcompound; toxin; auristatin; MMAE (CH₂)₁₅ S —(CH₂)₁₋₆— Cleavable ornon-cleavable linker; Spacer system Large, charged or hydrophobic di- ortri-or oligo peptide; val-cit; or absent compound; toxin; auristatin;MMAE (CH₂)₅ S —(CH₂)₁₀₋₂₀— Cleavable or non-cleavable linker; Spacersystem Large, charged or hydrophobic di- or tri-or oligo peptide;val-cit; or absent compound; toxin; auristatin; MMAE (CH₂)₁₀ S—(CH₂)₁₀₋₂₀— Cleavable or non-cleavable linker; Spacer system Large,charged or hydrophobic di- or tri-or oligo peptide; val-cit; or absentcompound; toxin; auristatin; MMAE (CH₂)₁₅ S —(CH₂)₁₀₋₂₀— Cleavable ornon-cleavable linker; Spacer system Large, charged or hydrophobic di- ortri-or oligo peptide; val-cit; or absent compound; toxin; auristatin;MMAE (CH₂)₅ S —(C═O)—O—CH₂— Cleavable or non-cleavable linker; Spacersystem Large, charged or hydrophobic di- or tri-or oligo peptide;val-cit; or absent compound; toxin; auristatin; MMAE (CH₂)₁₀ S—(C═O)—O—CH₂— Cleavable or non-cleavable linker; Spacer system Large,charged or hydrophobic di- or tri-or oligo peptide; val-cit; or absentcompound; toxin; auristatin; MMAE (CH₂)₁₅ S —(C═O)—O—CH₂— Cleavable ornon-cleavable linker; Spacer system Large, charged or hydrophobic di- ortri-or oligo peptide; val-cit; or absent compound; toxin; auristatin;MMAE (CH₂)₅ S —(C═O)—O—(CH₂)₂₋₂₀— Cleavable or non-cleavable linker;Spacer system Large, charged or hydrophobic di- or tri-or oligo peptide;val-cit; or absent compound; toxin; auristatin; MMAE (CH₂)₁₀ S—(C═O)—O—(CH₂)₂₋₂₀— Cleavable or non-cleavable linker; Spacer systemLarge, charged or hydrophobic di- or tri-or oligo peptide; val-cit; orabsent compound; toxin; auristatin; MMAE (CH₂)₁₅ S —(C═O)—O—(CH₂)₂₋₂₀—Cleavable or non-cleavable linker; Spacer system Large, charged orhydrophobic di- or tri-or oligo peptide; val-cit; or absent compound;toxin; auristatin; MMAE (CH₂)₅ S Amino acid, di- or tri-or Cleavable ornon-cleavable linker; Spacer system Large, charged or hydrophobic oligopeptide di- or tri-or oligo peptide; val-cit; or absent compound; toxin;auristatin; MMAE (CH₂)₁₀ S Amino acid, di- or tri-or Cleavable ornon-cleavable linker; Spacer system Large, charged or hydrophobic oligopeptide di- or tri-or oligo peptide; val-cit; or absent compound; toxin;auristatin; MMAE (CH₂)₁₅ S Amino acid, di- or tri-or Cleavable ornon-cleavable linker; Spacer system Large, charged or hydrophobic oligopeptide di- or tri-or oligo peptide; val-cit; or absent compound; toxin;auristatin; MMAE (CH₂)₅ S

Cleavable or non-cleavable linker; di- or tri-or oligo peptide; val-cit;or absent Spacer system; CH₂—(CH₂—O—CH₂)₄—CH₂— Large, charged orhydrophobic compound; toxin, auristatin; MMAE (CH₂)₅ S

Cleavable or non-cleavable linker; di- or tri-or oligo peptide; val-cit;or absent Spacer system; CH₂—(CH₂—O—CH₂)₄—CH₂— Large, charged orhydrophobic compound; toxin; auristatin; MMAE (CH₂)₅ S

Cleavable or non-cleavable linker; di- or tri-or oligo peptide; val-cit;or absent Spacer system; CH₂—(CH₂—O—CH₂)₄—CH₂— Large, charged orhydrophobic compound; toxin; auristatin; MMAE —CH₂—(CH₂—O—CH₂)₃—CH₂— S —Cleavable or non-cleavable linker; Spacer system Large, charged orhydrophobic di- or tri-or oligo peptide; val-cit; or absent compound;toxn; auristatin; MMAE —CH₂—(CH₂—O—CH₂)₃—CH₂— S —CH₂—(CH₂O—CH₂)₁₋₂₄—CH₂—Cleavable or non-cleavable linker, Spacer system Large, charged orhydrophobic di- or tri-or oligo peptide; val-cit; or absent compound;toxic, auristatin; MMAE —CH₂—(CH₂—O—CH₂)₃—CH₂— S—(C═O)—CH₂—(CH₂—O—CH₂)₁₋₂₄—CH₂— Cleavable or non-cleavable linker;Spacer system Large, charged or hydrophobic di- or tri-or oligo peptide;val-cit; or absent compound; toxin; auristatin; MMAE —O—(CH₂)₁₋₅ S —Cleavable or non-cleavable linker; Spacer system Large, charged orhydrophobic di- or tri-or oligo peptide; val-cit; or absent compound;toxin; auristatin; MMAE —O—CH₂—(CH₂—O—CH₂)₃—CH₂— S — Cleavable ornon-cleavable linker; Spacer system Large, charged or hydrophobic di- ortri-or oligo peptide; val-cit; or absent compound; toxin; auristatin;MMAE —O—CH₂—(CH₂—O—CH₂)₃—CH₂— S —CH₂—(CH₂—O—CH₂)₁₋₂₄—CH₂— Cleavable ornon-cleavable linker; Spacer system Large, charged or hydrophobic di- ortri-or oligo peptide; val-cit; or absent compound; toxin; auristatin;MMAE —O—CH₂—(CH₂—O—CH₂)₃—CH₂— S —(C—O)—CH₂—(CH₂—O—CH₂)₁₋₂₄—CH₂—Cleavable or non-cleavable linker; Spacer system Large, charged orhydrophobic di- or tri-or oligo peptide; val-cit; or absent compound;toxin; auristatin; MMAE (CH₂)₄—CH(NH₂)—(C═O)— S — — Charged compound(CH₂)₄—CH(NH₂)—(C═O)— S — di- or tri-or oligo peptide; val-cit Spacersystem Large, charged or hydrophobic compound; toxin; auristatin; MMAE(CH₂)₄—CH(NH₂)—(C═O)— S —(CH₂)₁₋₆— Cleavable or non-cleavable linker;Spacer system Large, charged or hydrophobic di- or tri-or oligo peptide;val-cit; or absent compound; toxin; auristatin; MMAE(CH₂)₄—CH(NH₂)—(C═O)— S —(CH₂)₅— Cleavable or non-cleavable linker;Spacer system Large, charged or hydrophobic di- or tri-or oligo peptide;val-cit; or absent compound; toxin; auristatin; MMAE(CH₂)₄—CH(NH₂)—(C═O)— S —CH₂—(CH₂—O—CH₂)₁₋₂₄—CH₂— Cleavable ornon-cleavable linker; Spacer system Large, charged or hydrophobic di- ortri-or oligo peptide; val-cit; or absent compound; toxin; auristatin;MMAF (CH₂)₅ S — — Spacer system; Large, charged or hydrophobic (CH₂)₅compound; toxin; auristatin; MMAF (CH₂)₆₋₁₀ S — — Spacer system; Large,charged or hydrophobic (CH₂)₅ compound; toxin; auristain; MMAF (CH₂)₁₅ S— — Spacer system; Large, charged or hydrophobic (CH₂)₅ compound; toxin;auristatin; MMAF —CH₂—(CH₂—O—CH₂)₃—CH₂— S — — Spacer system; Large,charged or hydrophobic (CH₂)₃ compound; toxin; auristatin; MMAE (CH₂)₅ S—(C═O)—CH₂— — Spacer system; Large, charged or hydrophobic (CH₂)₅compound; toxin; auristatin; MMAF (CH₂)₅ S —(C═O)—(CH₂)₅— — Spacersystem; Large, charged or hydrophobic (CH₂)₅ compound; toxin;auristatin; MMAF (CH₂)₅ S —CH₂—(CH₂—O—CH₂)₁₋₂₄—CH₂— — Spacer system;Large, charged or hydrophobic (CH₂)₅ compound; toxin; auristatin; MMAF—O—(CH₂)₁₋₅ S —CH₂—(CH₂—O—CH₂)₁₋₂₄—CH₂— — Spacer system; Large, chargedor hydrophobic (CH₂)₅ compound; toxin; auristatin; MMAF—O—CH₂—(CH₂—O—CH₂)₃—CH₂— S —CH₂—(CH₂—O—CH₂)₁₋₂₄—CH₂— — Spacer system;Large, charged or hydrophobic (CH₂)₅ compound; toxin; auristatin; MMAF—O—CH₂—(CH₂—O—CH₂)₃—CH₂— S —(C═O)—CH₂— — Spacer system, Large, chargedor hydrophobic (CH₂)₅ compound; toxin; auristatin; MMAF—O—CH₂—(CH₂—O—CH₂)₃—CH₂— S —(C═O)—(CH₂)₅— — Spacer system; Large,charged or hydrophobic (CH₂)₅ compound; toxin; auristatin; MMAF—CH₂—(CH₂—O—CH₂)₃—CH₂— S —CH₂—(CH₂O—CH₂)₁₋₂₄—CH₂— — Spacer system;Large, charged or hydrophobic (CH₂)₅ compound; toxin; auristatin; MMAF(CH₂)₅ S — Non-cleavable linker; (CH₂)₅ — Large, charged or hydrophobiccompound; toxin; auristatin; MMAF (CH₂)₆₋₁₀ S — Non-cleavable linker;(CH₂)₅ — Large, charged or hydrophobic compound; toxin; auristatin; MMAF(CH₂)₁₅ S — Non-cleavable linker; (CH₂)₅ — Large, charged or hydrophobiccompound; toxin; auristatin; MMAF —CH₂—(CH₂—O—CH₂)₃—CH₂— S —Non-cleavable linker; (CH₂)₅ — Large, charged or hydrophobic compound;toxin; auristatin; MMAF (CH₂)₅ S —(C═O)—CH₂— Non-cleavable linker;(CH₂)₅ — Large, charged or hydrophobic compound; toxin; auristatin; MMAF(CH₂)₅ S —(C═O)—(CH₂)₅— Non-cleavable linker; (CH₂)₅ — Large, charged orhydrophobic compound; toxin; auristatin; MMAF (CH₂)₅ S —(C═O)—(CH₂)₅—Non-cleavable linker; (CH₂)₅ — Large, charged or hydrophobic compound;toxin; auristatin; MMAF (CH₂)₅ S —(C═O)—(CH₂)₅— Non-cleavable linker;(CH₂)₅ — Large, charged or hydrophobic compound; toxin; auristatin; MMAF(CH₂)₅ S —CH₂—(CH₂—O—CH₂)₁₋₂₄—CH₂— Non-cleavable linker; (CH₂)₅ — Large,charged or hydrophobic compound; toxin; auristatin; MMAF —O—(CH₂)₁₋₅ S—CH₂—(CH₂—O—CH₂)₁₋₂₄—CH₂— Non-cleavable linker; (CH₂)₅ — Large, chargedor hydrophobic compound; toxin; auristatic; MMAF—O—CH₂—(CH₂—O—CH₂)₃—CH₂— S —CH₂—(CH₂—O—CH₂)₁₋₂₄—CH₂— Non-cleavablelinker; (CH₂)₅ — Large, charged or hydrophobic compound; toxin;auristatin; MMAF —O—CH₂—(CH₂—O—CH₂)₃—CH₂— S —(C═O)—CH₂— Non-cleavablelinker; (CH₂)₅ — Large, charged or hydrophobic compound; toxin;auristatin; MMAF —O—CH₂—(CH₂—O—CH₂)₃—CH₂— S —(C═O)—(CH₂)₅— Non-cleavablelinker; (CH₂)₅ — Large, charged or hydrophobic compound; toxin;auristatin; MMAF —CH₂—(CH₂—O—CH₂)₃—CH₂— S —CH₂—(CH₂—O—CH₂)₁₋₂₄—CH₂—Non-cleavable linker; (CH₂)₅ — Large, charged or hydrophobic compound;toxin; auristatin; MMAF (CH₂)₅ — —(C═O)—CH₂— — — Charged compound (CH₂)₅— —(C═O)—(CH₂)₅— — Spacer system Large, charged or hydrophobic compound;toxin; auristatin; MMAF (CH₂)₅ — —(C═O)—(CH₂)₅— Cleavable ornon-cleavable linker; Spacer system Large, charged or hydrophobic di- ortri-or oligo peptide; val-cit compound; toxin; auristatin; MMAE (CH₂)₅ ——(C═O)—(CH₂)₅— Cleavable or non-cleavable linker; Spacer system Large,charged or hydrophobic di- or tri-or oligo peptide; val-cit; or absentcompound; toxin; auristatin; MMAE (CH₂)₁₀ — —(C═O)—(CH₂)₅— Cleavable ornon-cleavable linker; Spacer system Large, charged or hydrophobic di- orti-or oligo peptide; val-cit; or absent compound; toxin; auristatin;MMAE (CH₂)₁₅ — —(C═O)—(CH₂)₅— Cleavable or non-cleavable linker; Spacersystem Large, charged or hydrophobic di- or tri-or oligo peptide;val-cit; or absent compound; toxin; auristatin; MMAE (CH₂)₅ ——CH₂—(CH₂—O—CH₂)₄—CH₂— Cleavable or non-cleavable linker; Spacer systemLarge, charged or hydrophobic di- or ti-or oligo peptide; val-cit; orabsent compound; toxin; auristatin; MMAE (CH₂)₅ ——CH₂—(CH₂—O—CH₂)₁₂—CH₂— Cleavable or non-cleavable linker; Spacer systemLarge, charged or hydrophobic di- or tri-or oligo peptide; val-cit; orabsent compound; toxin; auristatin; MMAE (CH₂)₅ ——CH₂—(CH₂—O—CH₂)₁₋₂₄—CH₂— Cleavable or non-cleavable linker; Spacersystem Large, charged or hydrophobic di- or tri-or oligo peptide;val-cit; or absent compound; toxin; auristatin; MMAE (CH₂)₁₀ ——CH₂—(CH₂—O—CH₂)₄—CH₂— Cleavable or non-cleavable linker; Spacer systemLarge, charged or hydrophobic di- or tri-or oligo peptide; val-cit; orabsent compound; toxin; auristatin; MMAE (CH₂)₁₅ ——CH₂—(CH₂—O—CH₂)₄—CH₂— Cleavable or non-cleavable linker; Spacer systemLarge, charged or hydrophobic di- or tri-or oligo peptide; val-cit; orabsent compound; toxin; auristatin; MMAE (CH₂)₅ ——(C═O)—CH₂—(CH₂—O—CH₂)₄—CH₂— Cleavable or non-cleavable linker; Spacersystem Large, charged or hydrophobic di- or tri-or oligo peptide;val-cit; or absent compound; toxin; auristatin; MMAE (CH₂)₁₀ ——(C═O)—CH₂—(CH₂—O—CH₂)₁₋₂₄—CH₂— Cleavable or non-cleavable linker;Spacer system Large, charged or hydrophobic di- or tri-or oligo peptide;val-cit; or absent compound; toxin; auristatin; MMAE (CH₂)₁₅ ——(C═O)—CH₂—(CH₂—O—CH₂)₁₋₂₄—CH₂— Cleavable or non-cleavable linker;Spacer system Large, charged or hydrophobic di- or tri-or oligo peptide;val-cit; or absent compound; toxin; auristatin; MMAE (CH₂)₅ ——(C═O)—(CH₂)₁₀₋₂₀— Cleavable or non-cleavable linker; Spacer systemLarge, charged or hydrophobic di- or tri-or oligo peptide; val-cit; orabsent compound; toxin; auristatin; MMAE (CH₂)₁₀ — —(C═O)—(CH₂)₁₀₋₂₀—Cleavable or non-cleavable linker; Spacer system Large, charged orhydrophobic di- or tri-or oligo peptide; val-cit; or absent compound;toxin; auristatin; MMAE (CH₂)₁₅ — —(C═O)—(CH₂)₁₀₋₂₀— Cleavable ornon-cleavable linker; Spacer system Large, charged or hydrophobic di- ortri-or oligo peptide; val-cit; or absent compound; toxin; auristatin;MMAE (CH₂)₅ — —(CH₂)₁₋₆— Cleavable or non-cleavable linker; Spacersystem Large, charged or hydrophobic di- or tri-or oligo peptide;val-cit; or absent compound; toxin; auristatin; MMAE (CH₂)₁₀ ——(CH₂)₁₋₆— Cleavable or non-cleavable linker; Spacer system Large,charged or hydrophobic di- or tri-or oligo peptide; val-cit; or absentcompound; toxin; auristatin; MMAE (CH₂)₁₅ — —(CH₂)₁₋₆— Cleavable ornon-cleavable linker; Spacer system Large, charged or hydrophobic di- ortri-or oligo peptide; val-cit; or absent compound; toxin; auristatin;MMAE (CH₂)₅ — —(CH₂)₁₀₋₂₀— Cleavable or non-cleavable linker; Spacersystem Large, charged or hydrophobic di- or tri-or oligo peptide;val-cit; or absent compound; toxin; auristatin; MMAE (CH₂)₁₀ ——(CH₂)₁₀₋₂₀— Cleavable or non-cleavable linker; Spacer system Large,charged or hydrophobic di- or tri-or oligo peptide; val-cit; or absentcompound; toxin; auristatin; MMAE (CH₂)₁₅ — —(CH₂)₁₀₋₂₀— Cleavable ornon-cleavable linker; Spacer system Large, charged or hydrophobic di- ortri-or oligo peptide; val-cit; or absent compound; toxin; auristatin;MMAE (CH₂)₅ — —(C═O)—O—CH₂— Cleavable or non-cleavable linker; Spacersystem Large, charged or hydrophobic di- or tri-or oligo peptide;val-cit; or absent compound; toxin; auristatin; MMAE (CH₂)₁₀ ——(C═O)—O—CH₂— Cleavable or non-cleavable linker; Spacer system Large,charged or hydrophobic di- or tri-or oligo peptide; val-cit; or absentcompound; toxin; auristatin; MMAE (CH₂)₁₅ — —(C═O)—O—CH₂— Cleavable ornon-cleavable linker; Spacer system Large, charged or hydrophobic di- ortri-or oligo peptide; val-cit; or absent compound; toxin; auristatin;MMAE (CH₂)₅ — —(C═O)—O—(CH₂)₂₋₂₀— Cleavable or non-cleavable linker;Spacer system Large, charged or hydrophobic di- or tri-or oligo peptide;val-cit; or absent compound; toxin; auristatin; MMAE (CH₂)₁₀ ——(C—O)—O—(CH₂)₂₋₂₀— Cleavable or non-cleavable linker; Spacer systemLarge, charged or hydrophobic di- or tri-or oligo peptide; val-cit; orabsent compound; toxin; auristatin; MMAE (CH₂)₁₅ — —(C═O)—O—(CH₂)₂₋₂₀—Cleavable or non-cleavable linker; Spacer system Large, charged orhydrophobic di- or tri-or oligo peptide; val-cit; or absent compound;toxin; auristatin; MMAE (CH₂)₅ — Amino acid, di- or tri-or Cleavable ornon-cleavable linker; Spacer system Large, charged or hydrophobic oligopeptide di- or tri-or oligo peptide; val-cit; or absent compound; toxin;auristatin; MMAE (CH₂)₁₀ — Amino acid, di- or tri-or Cleavable ornon-cleavable linker; Spacer system Large, charged or hydrophobic oligopeptide di- o rtri-or oligo peptide; val-cit; or absent compound; toxin;auristatin; MMAE (CH₂)₁₅ — Amino acid, di- or tri-or Cleavable ornon-cleavable linker; Spacer system Large, chargedo rhydophobic oligopeptide di- or tri-or oligo peptide; val-cit; or absent compound; toxin;auristatin; MMAE (CH₂)₅ —

Cleavable or non-cleavable linker; di- or tri-or oligo peptide; val-cit;or absent Spacer system; CH₂—(CH₂—O—CH₂)₄—CH₂— Large, charged orhydrophobic compound; toxin; auristatin; MMAE (CH₂)₅ —

Cleavable or non-cleavable linker; di- or tri-or oligo peptide; val-cit;or absent Spacer system; CH₂—(CH₂—O—CH₂)₄—CH₂— Large, charged orhydrophobic compound; toxin; auristatin; MMAE (CH₂)₅ —

Cleavable or non-cleavable linker; di- or tri-or oligo peptide; val-cit;or absent Spacer system; CH₂—(CH₂—O—CH₂)₄—CH₂— Large, charged orhydrophobic compound; toxin; auristatin; MMAE —CH₂—(CH₂—O—CH₂)₃—CH₂— — —Cleavable or non-cleavable linker; Spacer system Large, charged orhydrophobic di- or tri-or oligo peptide; val-cit; or absent compound;toxin; auristatin; MMAE —CH₂—(CH₂—O—CH₂)₃—CH₂— ——CH₂—(CH₂—O—CH₂)₁₋₂₄—CH₂— Cleavable or non-cleavable linker; Spacersystem Large, charged or hydrophobic di- or tri-or oligo peptide;val-cit; or absent compound; toxin; auristatin; MMAE—CH₂—(CH₂—O—CH₂)₃—CH₂— — —(C═O)—CH₂—(CH₂—O—CH₂)₁₋₂₄—CH₂— Cleavable ornon-cleavable linker; Spacer system Large, charged or hydrophobic di- ortri-or oligo peptide; val-cit; or absent compound; toxin; auristatin;MMAE —O—(CH₂)₁₋₅ — — Cleavable or non-cleavable linker; Spacer systemLarge, charged or hydrophobic di- or tri-or oligo peptide; val-cit; orabsent compound; toxin; auristatin; MMAE —O—CH₂—(CH₂—O—CH₂)₃—CH₂— — —Cleavable or non-cleavable linker; Spacer system Large, charged orhydrophobic di- or tri-or oligo peptide; val-cit; or absent compound;toxin; auristatin; MMAE —O—CH₂—(CH₂—O—CH₂)₃—CH₂— ——CH₂—(CH₂—O—CH₂)₁₋₂₄—CH₂— Cleavable or non-cleavable linker; Spacersystem Large, charged or hydrophobic di- or tri-or oligo peptide;val-cit; or absent compound; toxin; auristatin; MMAE—O—CH₂—(CH₂—O—(CH₂)₃—CH₂— — —(C═O)—CH₂—(CH₂—O—CH₂)₁₋₂₄—CH₂— Cleavable ornon-cleavable linker; Spacer system Large, charged or hydrophobic di- ortri-or oligo peptide; val-cit; or absent compound; toxin; auristatin;MMAE (CH₂)₄—CH(NH₂)—(C═O)— — — — Charged compound (CH₂)₄—CH(NH₂)—(C═O)—— — di- or tri-or oligo peptide; val-cit Spacer system Large, charged orhydrophobic compound; toxin; auristatin; MMAE (CH₂)₄—CH(NH₂)—(C═O)— ——(CH₂)₁₋₆— Cleavable or non-cleavable linker; Spacer system Large,charged or hydrophobic di- or tri-or oligo peptide; val-cit; or absentcompound; toxin; auristatin; MMAE (CH₂)₄—CH(NH₂)—(C═O)— — —(CH₂)₅—Cleavable or non-cleavable linker; Spacer system Large, charged orhydrophobic di- or tri-or oligo peptide; val-cit; or absent compound;toxin; auristatin; MMAF (CH₂)₄—CH(NH₂)—(C═O)— ——CH₂—(CH₂—O—CH₂)₁₋₂₄—CH₂— Cleavable or non-cleavable linker; Spacersystem Large, charged or hydrophobic di- or tri-or oligo peptide;val-cit; or absent compound; toxin; auristatin; MMAE (CH₂)₅ — — — Spacersystem; Large, charged or hydrophobic (CH₂)₅ compound; toxin;auristatin; MMAF (CH₂)₆₋₁₀ — — — Spacer system; Large, charged orhydrophobic (CH₂)₅ compound; toxin; auristatin; MMAF (CH₂)₁₅ — — —Spacer system; Large; charged or hydrophobic (CH₂)₅ compound; toxin;auristatin; MMAF —CH₂—(CH₂—O—CH₂)₃—CH₂— — — — Spacer system; Large,charged or hydrophobic (CH₂)₅ compound; toxin; auristatin; MMAF (CH₂)₅ ——(C═O)—CH₂— — Spacer system; Large, charged or hydrophobic (CH₂)₅compound; toxin; auristatin; MMAF (CH₂)₅ — —(C═O)—(CH₂)₅— — Spacersystem; Large, charged or hydrophobic (CH₂)₅ compound; toxin;auristatin; MMAF (CH₂)₅ — —CH₂—(CH₂—O—CH₂)₁₋₂₄—CH₂— — Spacer system;Large, charged or hydrophobic (CH₂)₅ compound; toxin; auristatin; MMAF—O—(CH₂)₁₋₅ — —CH₂—(CH₂—O—CH₂)₁₋₂₄—CH₂— — Spacer system; Large, chargedor hydrophobic (CH₂)₅ compound; toxin; auristatin; MMAF—O—CH₂—(CH₂—O—CH₂)₃—CH₂— — —CH₂—(CH₂—O—CH₂)₁₋₂₄—CH₂— — Spacer system;Large, charged or hydrophobic (CH₂)₅ compound; toxin; auristatin; MMAF—O—CH₂—(CH₂—O—CH₂)₃—CH₂— — —(C═O)—CH₂— — Spacer system; Large, chargedor hydrophobic (CH₂)₅ compound; toxin; auristatin; MMAF—O—CH₂—(CH₂—O—CH₂)₃—CH₂— — —(C═O)—(CH₂)₅— — Spacer system; Large,charged or hydrophobic (CH₂)₅ compound; toxin; auristatin; MMAF—CH₂—(CH₂—O—CH₂)₃—CH₂— — —CH₂—(CH₂—O—CH₂)₁₋₂₄—CH₂— — Spacer system;Large, charged or hydrophobic (CH₂)₅ compound; toxin; auristatin; MMAF(CH₂)₅ — — Non-cleavable linker; (CH₂)₅ — Large, charged or hydrophobiccompound; toxin; auristatin; MMAF (CH₂)₆₋₁₀ — — Non-cleavable linker;(CH₂)₅ — Large, charged or hydrophobic compound; toxin; auristatin; MMAF(CH₂)₁₅ — — Non-cleavable linker; (CH₂)₅ — Large, charged or hydrophobiccompound; toxin; auristatin; MMAF —CH₂—(CH₂—O—CH₂)₃—CH₂— — —Non-cleavable linker; (CH₂)₅ — Large, charged or hydrophobic compound;toxin; auristatin; MMAF (CH₂)₅ — —(C═O)—CH₂— Non-cleavable linker;(CH₂)₅ — Large, charged or hydrophobic compound; toxin; auristatin; MMAF(CH₂)₅ — —(C═O)—(CH₂)₅— Non-cleavable linker; (CH₂)₅ — Large, charged orhydrophobic compound; toxin; auristatin; MMAF (CH₂)₅ — —(C═O)—(CH₂)₅—Non-cleavable linker; (CH₂)₅ — Large, charged or hydrophobic compound;toxin; auristatin; MMAF (CH₂)₅ — —(C═O)—(CH₂)₅— Non-cleavable linker;(CH₂)₅ — Large, charged or hydrophobic compound; toxin; auristatin; MMAF(CH₂)₅ — —CH₂—(CH₂—O—CH₂)₁₋₂₄—CH₂— Non-cleavable linker; (CH₂)₅ — Large,charged or hydrophobic compound; toxin; auristatin; MMAF —O—(CH₂)₁₋₅ ——CH₂—(CH₂—O—CH₂)₁₋₂₄—CH₂— Non-cleavable linker; (CH₂)₅ — Large, chargedor hydrophobic compound; toxin; auristatin; MMAF—O—CH₂—(CH₂—O—CH₂)₃—CH₂— — —CH₂—(CH₂—O—CH₂)₁₋₂₄—CH₂— Non-cleavablelinker; (CH₂) — Large, charged or hydrophobic compound; toxin;auristatin; MMAF —O—CH₂—(CH₂—O—CH₂)₃—CH₂— — —(C═O)—CH₂— Non-cleavablelinker (CH₂)₅ — Large, charged or hydrophobic compound; toxin;auristatin; MMAF —O—CH₂—(CH₂—O—CH₂)₃—CH₂— — —(C═O)—(CH₂)₅— Non-cleavablelinker; (CH₂)₅ — Large, charged or hydrophobic compound; toxin;auristatin; MMAF —CH₂—(CH₂—O—CH₂)₃—CH₂— — —CH₂—(CH₂—O—CH₂)₁₋₂₄—CH₂—Non-cleavable linker; (CH₂)₅ — Large, charged or hydrophobic compound;toxin; auristatin; MMAF

TABLE 3 Structure of Formula Ib (C)_(n) X

(CH₂)₅ NH (CH₂)₄—CH(NH₂)—(C═O)— —

(CH₂)₅ NH (CH₂)₅ NH (CH₂)₄—CH(NH₂)—(C═O)— — O—(CH₂)₅ NH O—(CH₂)₅ NH

(CH₂)₅ NH (CH₂)₅ NH (CH₂)₅ NH O—(CH₂)₅ NH

—CH₂—(CH₂—O—CH₂)₃—CH₂— — —CH₂—(CH₂—O—CH₂)₁₋₆—CH₂— —

(CH₂)₅ NH (CH₂)₅ NH O—(CH₂)₅ NH

(CH₂)₅ NH O—(CH₂)₅ NH —CH₂—(CH₂—O—CH₂)₆—CH₂— —

(CH₂)₅ NH —CH₂—(CH₂—O—CH₂)₆—CH₂— NH O—(CH₂)₅ NH

(CH₂)₅ NH —O—(CH₂)₅ NH —CH₂—(CH₂—O—CH₂)₆—CH₂— NH (CH₂)₅ NH (CH₂)₅ NH(CH₂)₄—CH(NH₂)—(C═O)— — —O—(CH₂)₅ NH (CH₂)₅ NH (CH₂)₅ NH(CH₂)₄—CH(NH₂)—(C═O)— — O—(CH₂)₅ NH —CH₂—(CH₂—O—CH₂)₄—CH₂— — (CH₂)₅ NH(CH₂)₅ NH (CH₂)₅ NH (CH₂)₄—CH(NH₂)—(C═O)— — O—(CH₂)₅ NH—CH₂—(CH₂—O—CH₂)₃—CH₂— — Structure of Formula Ib L V Y

—(C═O)—CH₂— — — —CH₂— — —

—(C═O)—(CH₂)₅— — — —(CH₂)₅— — — —(CH₂)₅— — — —(C═O)—(CH₂)₅— — ——(C═O)—(CH₂)₁₀— — —

—(C═O)—CH₂—(CH₂—O—CH₂)₄—CH₂— — — —(C═O)—CH₂—(CH₂—O—CH₂)₁₋₂₄—CH₂— — ——CH₂—(CH₂—O—CH₂)₁₋₂₄—CH₂— — — —(C═O)—CH₂—(CH₂—O—CH₂)₁₋₂₄—CH₂— — —

— — — —

—(C═O)—CH₂—(CH₂—O—CH₂)₄—CH₂— —(C═O)—CH₂—(CH₂—O—CH₂)₁₋₂₄—CH₂——(C═O)—CH₂—(CH₂—O—CH₂)₁₋₂₄—CH₂—

—(C═O)— — — (CH₂)₅ — — — — —

—(C═O)— — — — — — — — —

— — — — — — — — — —(C═O)—(CH₂)₅— — — —(CH₂)₅— — — —CH₂— — ——(C═O)—(CH₂)₅— — — —(C═O)—(CH₂)₅— — — —(CH₂)₅— — — —CH₂— — ——(C═O)—(CH₂)₅— — — — — — —(CH₂)₂— — — —(C═O)—(CH₂)₅— — — —(CH₂)₅— — ——CH₂— — — —(C═O)—CH₂— — — — — — Structure of Formula Ib R

SH SH

SH SH SH SH SH

SH SH SH SH

N₃ N₃

N₃ N₃ N₃

TABLE 4 Structure of Formula III R′

N₃ N₃ N₃ Structure of Formula III L′

—(CH₂)₅—(C═O)— —CH₂—(CH₂—O—CH₂)₄—CH₂— (C═O)—

—(C═O)—(CH₂)4-(C═O)—NH—CH₂— (CH₂—O—CH₂)₄—CH₂—(C═O)——(C═O)—(CH₂)4-(C═O)—NH—CH₂— (CH₂—O—CH₂)₄—CH₂—(C═O)—

—(CH₂)₅—(C═O)— —CH₂—(CH₂—O—CH₂)₄—CH₂— (C═O)— —CH₂—(CH₂—O—CH₂)₄—CH₂—(C═O)—

—(CH₂)₅—(C═O)— —CH₂—(CH₂—O—CH₂)₄—CH₂— (C═O)— —CH₂—(CH₂—O—CH₂)₄—CH₂—(C═O)— Structure of Formula III V′ Y′ Z

Val-cit PAB MMAE Val-cit PAB MMAE

Val-cit PAB MMAE — — MMAF

Val-cit PAB MMAE Val-cit PAB MMAE — — MMAF

Val-cit PAB MMAE Val-cit PAB MMAE — — MMAE

TABLE 5 Compound of Formula 1b

Compound of Formula III

All headings and sub-headings are used herein for convenience only andshould not be construed as limiting the invention in any way. Anycombination of the above-described elements in all possible variationsthereof is encompassed by the invention unless otherwise indicatedherein or otherwise clearly contradicted by context. Recitation ofranges of values herein are merely intended to serve as a shorthandmethod of referring individually to each separate value falling withinthe range, unless otherwise indicated herein, and each separate value isincorporated into the specification as if it were individually recitedherein. Unless otherwise stated, all exact values provided herein arerepresentative of corresponding approximate values (e.g., all exactexemplary values provided with respect to a particular factor ormeasurement can be considered to also provide a correspondingapproximate measurement, modified by “about,” where appropriate).

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext.

The use of any and all examples, or exemplary language (e.g., “such as”)provided herein is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise indicated. No language in the specification should beconstrued as indicating any element is essential to the practice of theinvention unless as much is explicitly stated.

The citation and incorporation of patent documents herein is done forconvenience only and does not reflect any view of the validity,patentability and/or enforceability of such patent documents. Thedescription herein of any aspect or embodiment of the invention usingterms such as reference to an element or elements is intended to providesupport for a similar aspect or embodiment of the invention that“consists of,” “consists essentially of” or “substantially comprises”that particular element or elements, unless otherwise stated or clearlycontradicted by context (e.g., a composition described herein ascomprising a particular element should be understood as also describinga composition consisting of that element, unless otherwise stated orclearly contradicted by context).

This invention includes all modifications and equivalents of the subjectmatter recited in the aspects or claims presented herein to the maximumextent permitted by applicable law.

All publications and patent applications cited in this specification areherein incorporated by reference in their entireties as if eachindividual publication or patent application were specifically andindividually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

1. An antibody or antibody fragment comprising a functionalized acceptorglutamine residue, the functionalized acceptor glutamine residue havingFormula IVa,(Q)-NH—(C)_(n)—X-L-(V—(Y—(Z)_(z))_(q))_(q))_(r)  Formula IVa or apharmaceutically acceptable salt or solvate thereof, wherein: Q isglutamine residue present in an antibody or antibody fragment; (C)_(n)is a substituted or unsubstituted alkyl or heteroalkyl chain, optionallywherein any carbon of the chain is substituted with an alkoxy, hydroxyl,alkylcarbonyloxy, alkyl-S—, thiol, alkyl-C(O)S—, amine, alkylamine,amide, or alkylamide; n is an integer selected from among the range of 2to 20; X is NH, O, S, absent, or a bond; L is independently absent, abond or a continuation of a bond, or a carbon comprising framework of 5to 200 atoms substituted at one or more atoms; r is an integer selectedfrom among 1, 2, 3 or 4; q is an integer selected from among 1, 2, 3 or4; z is an integer selected from among 1, 2, 3 or 4; and V isindependently absent, a bond or a continuation of a bond, anon-cleavable moiety or a conditionally-cleavable moiety; Y isindependently absent, a bond or a continuation of a bond, or a spacersystem which is comprised of 1 or more spacers; and Z is a moiety thatimproves pharmacokinetic properties, a therapeutic moiety or adiagnostic moiety, wherein Z is an organic compound that is electricallynegatively charged, hydrophobic and/or that has a molecular weight of atleast 400 g/mol.
 2. The antibody of claim 1, wherein n is an integerselected from among the range of 10 to
 20. 3. The antibody of claim 1,wherein (C)_(n) is a heteroalkyl chain that comprises a (CH₂—CH₂—O—)_(x)group, wherein x is an integer selected from among the range of 1 to 6.4. The antibody of claim 1, wherein where at least one of L, V or Y arepresent.
 5. The antibody of claim 4, wherein n is an integer selectedfrom among the range of 2 to
 6. 6. The antibody of claim 1, wherein saidacceptor glutamine residue is flanked at position +2 by a non-asparticacid residue.
 7. An antibody or antibody fragment comprising an acceptorglutamine residue flanked at the +2 position by a non-aspartic acidresidue, wherein the acceptor glutamine residue is functionalized with acompound comprising a moiety-of-interest.
 8. The antibody of claim 7,wherein said amino acid residue at the +2 position is not a glutamine.9. The antibody of claim 7, wherein said functionalized acceptorglutamine residue is in an antibody heavy chain at position 295 (EUnumbering).
 10. The antibody of claim 9, wherein said antibody comprisesa N297X substitution, wherein X is any amino acid other than asparticacid, asparagine or glutamine.
 11. The antibody of claim 7, wherein saidmoiety-of-interest is covalently bound to the acceptor glutamine residuevia a linker comprising a NH—(C)_(n) group, wherein (C)_(n) is asubstituted or unsubstituted alkyl or heteroalkyl chain, wherein anycarbon of the chain is optionally substituted with an alkoxy, hydroxyl,alkylcarbonyloxy, alkyl-S—, thiol, alkyl-C(O)S—, amine, alkylamine,amide, or alkylamide; and n is an integer selected from among the rangeof 2 to
 20. 12. The antibody of claim 7, wherein the functionalizedacceptor glutamine residue has a structure of Formula IVa,(Q)-NH—(C)_(n)—X-L-(V—(Y—(Z)_(z))_(q))_(r)  Formula IVa or apharmaceutically acceptable salt or solvate thereof, wherein: Q isglutamine residue present in an antibody or antibody fragment; (C)_(n)is a substituted or unsubstituted alkyl or heteroalkyl chain, optionallywherein any carbon of the chain is substituted with an alkoxy, hydroxyl,alkylcarbonyloxy, alkyl-S—, thiol, alkyl-C(O)S—, amine, alkylamine,amide, or alkylamide; n is an integer selected from among the range of 2to 20; X is NH, O, S, absent, or a bond; L is independently absent, abond or a continuation of a bond, or a carbon comprising framework of 5to 200 atoms substituted at one or more atoms; r is an integer selectedfrom among 1, 2, 3 or 4; q is an integer selected from among 1, 2, 3 or4; z is an integer selected from among 1, 2, 3 or 4; and V isindependently absent, a bond or a continuation of a bond, anon-cleavable moiety or a conditionally-cleavable moiety; Y isindependently absent, a bond or a continuation of a bond, or a spacersystem which is comprised of 1 or more spacers; and Z is a moiety thatimproves pharmacokinetic properties, a therapeutic moiety or adiagnostic moiety, wherein Z is an organic compound that is electricallynegatively charged, hydrophobic and/or that has a molecular weight of atleast 400 g/mol.
 13. An antibody or antibody fragment comprising afunctionalized acceptor glutamine residue, wherein said acceptorglutamine residue is flanked at the +2 position by a non-aspartic acidresidue, the functionalized acceptor glutamine residue having FormulaIVa,(Q)-NH—(C)_(n)—X-L-(V—(Y—(Z)_(z))_(q))_(r)  Formula IVa or apharmaceutically acceptable salt or solvate thereof, wherein: Q isglutamine residue present in an antibody or antibody fragment; (C)_(n)is a substituted or unsubstituted alkyl or heteroalkyl chain, optionallywherein any carbon of the chain is substituted with an alkoxy, hydroxyl,alkylcarbonyloxy, alkyl-S—, thiol, alkyl-C(O)S—, amine, alkylamine,amide, or alkylamide; n is an integer selected from among the range of 2to 20; X is NH, O, S, absent, or a bond; L is independently absent, abond or a continuation of a bond, or a carbon comprising framework of 5to 200 atoms substituted at one or more atoms, optionally, wherein thecarbon comprising framework comprises a linear framework of 5 to 30carbon atoms optionally substituted at one or more atoms, optionallywherein the carbon comprising framework is a linear hydrocarbon, asymmetrically or asymmetrically branched hydrocarbon, monosaccharide,disaccharide, linear or branched oligosaccharide (asymmetricallybranched or symmetrically branched), other natural linear or branchedoligomers (asymmetrically branched or symmetrically branched), or adimer, trimer, or higher oligomer (linear, asymmetrically branched orsymmetrically branched) resulting from any chain-growth or step-growthpolymerization process; r is an integer selected from among 1, 2, 3 or4; q is an integer selected from among 1, 2, 3 or 4; z is an integerselected from among 1, 2, 3 or 4; and V is independently absent, anon-cleavable moiety or a conditionally-cleavable moiety; Y isindependently absent, a bond or a continuation of a bond, or a spacersystem which is comprised of 1 or more spacers; and Z is a moiety thatimproves the pharmacokinetic properties, a therapeutic moiety or adiagnostic moiety.
 14. The antibody of claim 13, wherein Z is an organiccompound that is charged, hydrophobic and/or has a molecular weight ofat least 400 g/mol.
 15. The antibody of claim 13, wherein said aminoacid residue at the +2 position is not a glutamine.
 16. The antibody ofclaim 13, wherein said functionalized acceptor glutamine residue is inan antibody heavy chain at position 295 (EU numbering).
 17. The antibodyof claim 16, wherein said antibody comprises a N297X substitution,wherein X is any amino acid other than aspartic acid, asparagine orglutamine.
 18. An antibody or antibody fragment comprising afunctionalized acceptor glutamine residue having Formula II:(Q)-NH—(C)_(n)—X-L-(V—(Y—(R)_(z))_(q))_(r)  Formula II or apharmaceutically acceptable salt or solvate thereof, wherein: Q isglutamine residue present in an antibody or antibody fragment; (C)_(n)is a substituted or unsubstituted alkyl or heteroalkyl chain, optionallywherein any carbon of the chain is optionally substituted with alkoxy,hydroxyl, alkylcarbonyloxy, alkyl-S—, thiol, alkyl-C(O)S—, amine,alkylamine, amide, or alkylamide; n is an integer from among the rangeof 2 to 20; X is NH, O, S, absent, or a bond; L is independently absent,a bond or a continuation of a bond, or a carbon comprising framework of1 to 200 atoms substituted at one or more atoms, optionally wherein thecarbon comprising framework comprises a linear framework of 3 to 30carbon atoms optionally substituted at one or more atoms, optionallywherein the carbon comprising framework is a linear hydrocarbon, asymmetrically or asymmetrically branched hydrocarbon, monosaccharide,disaccharide, linear or branched oligosaccharide (asymmetricallybranched or symmetrically branched), other natural linear or branchedoligomers (asymmetrically branched or symmetrically branched), or adimer, trimer, or higher oligomer (linear, asymmetrically branched orsymmetrically branched) resulting from any chain-growth or step-growthpolymerization process; r is an integer selected from among 1, 2, 3 or4; q is an integer selected from among 1, 2, 3 or 4; z is an integerselected from among 1, 2, 3 or 4; and V is independently absent, a bondor a continuation of a bond, a non-cleavable moiety or aconditionally-cleavable moiety; Y is independently absent, a bond or acontinuation of a bond, or a spacer system which is comprised of 1 ormore spacers; and R is a reactive moiety.
 19. The antibody of claim 18,wherein R is a moiety comprising a bioorthogonal-reaction compatiblereactive group, for example an unprotected or protected thiol, epoxide,maleimide, haloacetamide, o-phoshenearomatic ester, azide, fulminate,sulfonate ester, alkyne, cyanide, amino-thiol, carbonyl, aldehyde,generally any group capable of oxime and hydrazine formation,1,2,4,5-tetrazine, norbornene, other stained or otherwise electronicallyactivated alkene, a substituted or unsubstituted cycloalkyne, generallyany reactive groups which form via bioorthogonal cycloaddition reactiona 1,3- or 1,5-disubstituted triazole, any diene or strained alkenedienophile that can react via inverse electron demand Diels-Alderreaction, a protected or unprotected amine, a carboxylic acid, analdehyde, an oxyamine.
 20. The antibody of claim 18, wherein saidacceptor glutamine residue is flanked at the +2 position by anon-aspartic acid residue.
 21. The antibody of claim 20, wherein saidnon-aspartic acid residue at the +2 position is not a glutamine.
 22. Theantibody of claim 18, wherein R is an azide.
 23. The antibody of claim18, wherein R comprises a cyclic group.
 24. The antibody of claim 18,wherein n is an integer from among the range of 10 to 20, and Rcomprises a cyclic group.
 25. An antibody or antibody fragmentcomprising a functionalized acceptor glutamine residue having FormulaIVb,(Q)-NH—(C)_(n)—X-L-(V—(Y-(M)_(z))_(q))_(r)  Formula IVb or apharmaceutically acceptable salt or solvate thereof, wherein: Q isglutamine residue present in an antibody or antibody fragment; (C)_(n)is a substituted or unsubstituted alkyl or heteroalkyl chain, optionallywherein any carbon of the chain is substituted with a alkoxy, hydroxyl,alkylcarbonyloxy, alkyl-S—, thiol, alkyl-C(O)S—, amine, alkylamine,amide, or alkylamide; n is an integer selected from among the range of 2to 20; X is NH, O, S, absent, or a bond; L is independently absent, abond or a continuation of a bond, or a carbon comprising framework of 1to 200 atoms substituted at one or more atoms, optionally, wherein thecarbon comprising framework comprises a linear framework of 3 to 30carbon atoms optionally substituted at one or more atoms, optionallywherein the carbon comprising framework is a linear hydrocarbon, asymmetrically or asymmetrically branched hydrocarbon, monosaccharide,disaccharide, linear or branched oligosaccharide (asymmetricallybranched or symmetrically branched), other natural linear or branchedoligomers (asymmetrically branched or symmetrically branched), or adimer, trimer, or higher oligomer (linear, asymmetrically branched orsymmetrically branched) resulting from any chain-growth or step-growthpolymerization process; r is an integer selected from among 1, 2, 3 or4; q is an integer selected from among 1, 2, 3 or 4; z is an integerselected from among 1, 2, 3 or 4; V is independently absent, a bond or acontinuation of a bond, a non-cleavable moiety or aconditionally-cleavable moiety; Y is independently absent, a bond or acontinuation of a bond, or a spacer system which is comprised of 1 ormore spacers; M is independently: R or(RR′)-L′-(V′—(Y′—(Z)_(z′))_(q′))_(r′), wherein R is a reactive moiety;(RR′) is an addition product between R and a complementary reactivemoiety R′; L′ is independently absent, a bond or a continuation of abond, or a carbon comprising framework of 1 to 200 atoms substituted atone or more atoms, optionally, wherein the carbon comprising frameworkcomprises a linear framework of 3 to 30 carbon atoms optionallysubstituted at one or more atoms, optionally wherein the carboncomprising framework is a linear hydrocarbon, a symmetrically orasymmetrically branched hydrocarbon, monosaccharide, disaccharide,linear or branched oligosaccharide (asymmetrically branched orsymmetrically branched), other natural linear or branched oligomers(asymmetrically branched or symmetrically branched), or a dimer, trimer,or higher oligomer (linear, asymmetrically branched or symmetricallybranched) resulting from any chain-growth or step-growth polymerizationprocess; V′ is independently absent, a bond or a continuation of a bond,a non-cleavable moiety or a conditionally-cleavable moiety; Y′ isindependently absent, a bond or a continuation of a bond, or a spacersystem which is comprised of 1 or more spacers; Z is independently areactive group, a moiety that improves the pharmacokinetic properties, atherapeutic or diagnostic moiety, and each Z is directly coupled toeither Y or V when Y is absent, or L when both Y and V are absent; andz′, q′ and r′ are each independently an integer selected from among 1,2, 3 or
 4. 26. The antibody of claim 25, wherein RR′ is a thio-maleimide(or halo-acetamide) addition, for example, aN,S-disubstituted-3-thio-pyrrolidine-2,5-dione; Staudinger ligation, forexample, a N,3- or N,4-substitued-5-dipenylphosphinoxide-benzoic amide;Huisgen 1,3-cycloaddition (click reaction), for example, aN,S-disubstituted-3-thio-pyrrolidine-2,5-dione,1,4-disubstituted-1,2,3-triazole, 3,5-disubstituted-isooxazole, or3,5-disubstituted-tetrazole; Diels-Alder cycloaddition adduct, forexample the 2,4-cycloaddition product between an O orN-substituted-5-norbornene-2-carboxylic ester or amide,N-substituted-5-norbornene-2,3-dicarboxylic imide, O orN-substituted-7-oxonorbornene-5-carboxylic ester or amide, orN-substituted-7-oxonorbornene-5,6-dicarboxylic imide and a 9-substitutedanthracene or 3-substituted 1,2,4,5-tetrazine; or any high yieldselective amidation or imidization reaction.
 27. The antibody of claim25, wherein said acceptor glutamine residue is flanked at the +2position by a non-aspartic acid residue.
 28. The antibody of claim 25,wherein L comprises a (CH₂—CH₂—O—)_(x) group, wherein x is an integerfrom among the range of 1 to
 24. 29. The antibody of claim 25, whereinthe groups —(C)_(n)—X-L-collectively comprise a structure(CH₂—CH₂—O—)_(x), wherein x is an integer from among the range of 3 to24.
 30. A composition comprising a plurality of antibodies of claim 1sharing the same heavy and/or light chain amino acid sequences, whereinat least 90% of the antibodies in said composition have (m)functionalized acceptor glutamine residues (Q) per antibody, wherein mis an integer selected from 1, 2, 3, or
 4. 31. The composition of claim30, wherein m is
 2. 32. A composition comprising a plurality ofantibodies of claim 25 sharing the same heavy and/or light chain aminoacid sequences, wherein at least 90% of the antibodies in saidcomposition have at least (m) functionalized acceptor glutamine residues(Q) per antibody, and wherein m is an integer selected from among 1, 2,3, or
 4. 33. The composition of claim 32, wherein m is
 4. 34. Acomposition comprising a plurality of antibodies comprising one acceptorglutamine on each heavy chain, wherein at least 80% of the antibodies inthe composition comprise on each heavy chain one functionalized acceptorglutamine residue (Q) having the structure of Formula IVa of claim 1.35. A composition comprising a plurality of antibodies comprising oneacceptor glutamine on each heavy chain, wherein at least 80% of theantibodies in the composition comprise on each heavy chain twofunctionalized acceptor glutamine residues (Q) having the structure ofFormula IVb of claim
 25. 36. A composition comprising a plurality ofantibodies linked to a moiety of interest (Z) via one functionalizedacceptor glutamine on each heavy chain of the antibody, wherein thecomposition is characterized by a mean Z:antibody ratio of at least 1.5,1.6, 1.7 or 1.8, wherein less than 10%, less than 5%, or less than 2% ofthe antibodies comprise more than two functionalized acceptor glutaminesper antibody.
 37. The composition of claim 36, wherein at least 80% ofthe antibodies in the composition comprise on each heavy chain onefunctionalized acceptor glutamine residue (Q) having the structure ofFormula IVa,(Q)-NH—(C)_(n)—X-L-(V—(Y—(Z)_(z))_(q))_(r)  Formula IVa or apharmaceutically acceptable salt or solvate thereof, wherein: Q isglutamine residue present in an antibody or antibody fragment; (C)_(n)is a substituted or unsubstituted alkyl or heteroalkyl chain, optionallywherein any carbon of the chain is substituted with an alkoxy, hydroxyl,alkylcarbonyloxy, alkyl-S—, thiol, alkyl-C(O)S—, amine, alkylamine,amide, or alkylamide; n is an integer selected from among the range of 2to 20; X is NH, O, S, absent, or a bond; L is independently absent, abond or a continuation of a bond, or a carbon comprising framework of 5to 200 atoms substituted at one or more atoms; r is an integer selectedfrom among 1, 2, 3 or 4; q is an integer selected from among 1, 2, 3 or4; z is an integer selected from among 1, 2, 3 or 4; and V isindependently absent, a bond or a continuation of a bond, anon-cleavable moiety or a conditionally-cleavable moiety; Y isindependently absent, a bond or a continuation of a bond, or a spacersystem which is comprised of 1 or more spacers; and Z is a moiety thatimproves pharmacokinetic properties, a therapeutic moiety or adiagnostic moiety, wherein Z is an organic compound that is electricallynegatively charged, hydrophobic and/or that has a molecular weight of atleast 400 g/mol.
 38. A composition comprising a plurality of antibodieslinked to a moiety of interest (Z) via two functionalized acceptorglutamines on each heavy chain of the antibody, wherein the compositionis characterized by a mean Z:antibody ratio of at least 3.2, 3.4, 3.5 or3.6, wherein less than 10%, less than 5%, or less than 2% of theantibodies comprise more than four functionalized acceptor glutaminesper antibody.
 39. The composition of claim 38, wherein at least 80% ofthe antibodies in the composition comprise on each heavy chain twofunctionalized acceptor glutamine residues (Q) having the structure ofFormula IVb,(Q)-NH—(C)_(n)—X-L-(V—(Y-(M)_(z))_(q))_(r)  Formula IVb or apharmaceutically acceptable salt or solvate thereof, wherein: Q isglutamine residue present in an antibody or antibody fragment; (C)_(n)is a substituted or unsubstituted alkyl or heteroalkyl chain, optionallywherein any carbon of the chain is substituted with a alkoxy, hydroxyl,alkylcarbonyloxy, alkyl-S—, thiol, alkyl-C(O)S—, amine, alkylamine,amide, or alkylamide; n is an integer selected from among the range of 2to 20; X is NH, O, S, absent, or a bond; L is independently absent, abond or a continuation of a bond, or a carbon comprising framework of 1to 200 atoms substituted at one or more atoms, optionally, wherein thecarbon comprising framework comprises a linear framework of 3 to 30carbon atoms optionally substituted at one or more atoms, optionallywherein the carbon comprising framework is a linear hydrocarbon, asymmetrically or asymmetrically branched hydrocarbon, monosaccharide,disaccharide, linear or branched oligosaccharide (asymmetricallybranched or symmetrically branched), other natural linear or branchedoligomers (asymmetrically branched or symmetrically branched), or adimer, trimer, or higher oligomer (linear, asymmetrically branched orsymmetrically branched) resulting from any chain-growth or step-growthpolymerization process; r is an integer selected from among 1, 2, 3 or4; q is an integer selected from among 1, 2, 3 or 4; z is an integerselected from among 1, 2, 3 or 4; V is independently absent, a bond or acontinuation of a bond, a non-cleavable moiety or aconditionally-cleavable moiety; Y is independently absent, a bond or acontinuation of a bond, or a spacer system which is comprised of 1 ormore spacers; M is independently: R or(RR′)-L′-(V′—(Y′—(Z)_(z′))_(q′))_(r′), wherein R is a reactive moiety;(RR′) is an addition product between R and a complementary reactivemoiety R′; L′ is independently absent, a bond or a continuation of abond, or a carbon comprising framework of 1 to 200 atoms substituted atone or more atoms, optionally, wherein the carbon comprising frameworkcomprises a linear framework of 3 to 30 carbon atoms optionallysubstituted at one or more atoms, optionally wherein the carboncomprising framework is a linear hydrocarbon, a symmetrically orasymmetrically branched hydrocarbon, monosaccharide, disaccharide,linear or branched oligosaccharide (asymmetrically branched orsymmetrically branched), other natural linear or branched oligomers(asymmetrically branched or symmetrically branched), or a dimer, trimer,or higher oligomer (linear, asymmetrically branched or symmetricallybranched) resulting from any chain-growth or step-growth polymerizationprocess; V′ is independently absent, a bond or a continuation of a bond,a non-cleavable moiety or a conditionally-cleavable moiety; Y′ isindependently absent, a bond or a continuation of a bond, or a spacersystem which is comprised of 1 or more spacers; Z is independently areactive group, a moiety that improves the pharmacokinetic properties, atherapeutic or diagnostic moiety, and each Z is directly coupled toeither Y or V when Y is absent, or L when both Y and V are absent; andz′, q′ and r′ are each independently an integer selected from among 1,2, 3 or
 4. 40. A linking reagent, a pharmaceutically acceptable salt orsolvate thereof, or a protein-conjugated linking reagent having thegeneral Formula Ib:G-NH—(C)_(n)—X-L-(V—(Y—(R)_(z))_(q))_(r)  Formula Ib or apharmaceutically acceptable salt or solvate thereof, wherein: G is a H,amine protecting group, or upon conjugation, an antibody or antibodyfragment attached via an amide bond; (C)_(n) is a substituted orunsubstituted alkyl or heteroalkyl chain, optionally wherein any carbonof the chain is substituted with an alkoxy, hydroxyl, alkylcarbonyloxy,alkyl-S—, thial, alkyl-C(O)S—, amine, alkylamine, amide, or alkylamide;n is an integer selected from among the range of 2 to 20; X is NH, O, S,absent or a bond; L is independently absent, a bond or a continuation ofa bond, or a carbon comprising framework of 1 to 200 atoms substitutedat one or more atoms, optionally, wherein the carbon comprisingframework comprises a linear framework of 5 to 30 atoms optionallysubstituted at one or more atoms, optionally wherein the carboncomprising framework is a linear hydrocarbon, a symmetrically orasymmetrically branched hydrocarbon, monosaccharide, disaccharide,linear or branched oligosaccharide (asymmetrically branched orsymmetrically branched), other natural linear or branched oligomers(asymmetrically branched or symmetrically branched), or a dimer, trimer,or higher oligomer (linear, asymmetrically branched or symmetricallybranched) resulting from any chain-growth or step-growth polymerizationprocess; r is an integer selected from among 1, 2, 3 or 4; q is aninteger selected from among 1, 2, 3 or 4; z is an integer selected fromamong 1, 2, 3 or 4; V is independently absent, a non-cleavable moiety ora conditionally-cleavable moiety, optionally following prior conditionaltransformation, which can be cleaved or transformed by a chemical,photochemical, physical, biological, or enzymatic process; Y isindependently absent, a bond or a continuation of a bond, or a spacersystem which is comprised of 1 or more spacers; and R is a reactivemoiety.
 41. A compound having the structure of Formula III, below,R′-L-(V—(Y-(M)_(z))_(q))_(r)  Formula III or a pharmaceuticallyacceptable salt or solvate thereof, wherein: R′ is a reactive group; Lis independently absent, or a carbon comprising framework of 1 to 200atoms substituted at one or more atoms, optionally, wherein the carboncomprising framework comprises a linear framework of 5 to 30 atomsoptionally substituted at one or more atoms, optionally wherein thecarbon comprising framework is a linear hydrocarbon, a symmetrically orasymmetrically branched hydrocarbon, monosaccharide, disaccharide,linear or branched oligosaccharide (asymmetrically branched orsymmetrically branched), other natural linear or branched oligomers(asymmetrically branched or symmetrically branched), or a dimer, trimer,or higher oligomer (linear, asymmetrically branched or symmetricallybranched) resulting from any chain-growth or step-growth polymerizationprocess; V is independently absent, a bond or a continuation of a bond,a non-cleavable moiety or a conditionally-cleavable moiety; Y isindependently absent, a bond or a continuation of a bond, or a spacersystem which is comprised of 1 or more spacers; M is independently: R or(RR′)-L′-(V′—(Y′—(Z)_(z′))_(q′))_(r′), wherein R is a reactive moiety;(RR′) is an addition product between an R and a complementary reactivegroup R′; L′ is independently absent, a bond or a continuation of abond, or a carbon comprising framework of 1 to 200 atoms substituted atone or more atoms, optionally, wherein the carbon comprising frameworkcomprises a linear framework of 3 to 30 carbon atoms optionallysubstituted at one or more atoms, optionally wherein the carboncomprising framework is a linear hydrocarbon, a symmetrically orasymmetrically branched hydrocarbon, monosaccharide, disaccharide,linear or branched oligosaccharide (asymmetrically branched orsymmetrically branched), other natural linear or branched oligomers(asymmetrically branched or symmetrically branched), or a dimer, trimer,or higher oligomer (linear, asymmetrically branched or symmetricallybranched) resulting from any chain-growth or step-growth polymerizationprocess; V′ is independently absent, a bond or a continuation of a bond,a non-cleavable moiety or a conditionally-cleavable moiety; Y′ isindependently absent, a bond or a continuation of a bond, or a spacersystem which is comprised of 1 or more spacers; Z is independently areactive group, a moiety that improves the pharmacokinetic properties, atherapeutic or diagnostic moiety, and each Z is directly coupled toeither Y or V when Y is absent, or L when both Y and V are absent; andz′, q′ and r′ are each independently an integer selected from among 1,2, 3 or
 4. 42. A method for conjugating a moiety of interest (Z) to anantibody, comprising the steps of: a) providing an antibody having atleast one acceptor glutamine residue; b) reacting said antibody with alinking reagent comprising a reactive group or protected reactive group(R) of claim 40, in the presence of a TGase, under conditions sufficientto obtain an antibody comprising an acceptor glutamine linked(covalently) to a reactive group (R) via a lysine-based linker; and (c)optionally, reacting (i) an antibody obtained in step b) with (ii) acompound comprising a moiety of interest (Z) and a reactive group (R′)capable of reacting with reactive group (R), under conditions sufficientto obtain an antibody comprising an acceptor glutamine linked to amoiety of interest (Z) via a lysine-based linker is obtained.
 43. Amethod for evaluating an antibody conjugate, the method comprising thesteps of: a) providing a first antibody composition of Formula IVa orFormula IVb comprising a first X, L, V, Y, L′, V′, Y′, (RR′) and/or Zmoiety, wherein at least 70%, 80% or 90% of the antibodies in said firstantibody composition have (m) functionalized acceptor glutamine residues(Q) per antibody, wherein m is an integer selected from 1, 2, 3, or 4;b) providing a second antibody composition of Formula IVa or Formula IVbcomprising a second X, L, V, Y, L′, V′, Y′, (RR′) and/or Z moiety,wherein said second antibody comprises at least one X, L, V, Y, L′, V′,Y′, (RR′) and/or Z moiety that differs from a respective X, L, V, Y, L′,V′, Y′, (RR′) and/or Z moiety of said first antibody, wherein at least70%, 80% or 90% of the antibodies in said second antibody compositionhave (n) functionalized acceptor glutamine residues (Q) per antibody,wherein n is an integer selected from 1, 2, 3, or 4; and c) evaluatingthe first and second antibody compositions; wherein Formula IVa is:(Q)-NH—(C)_(n)—X-L-(V—(Y—(Z)_(z))_(q))_(r)  Formula IVa or apharmaceutically acceptable salt or solvate thereof, wherein: Q isglutamine residue present in an antibody or antibody fragment; (C)_(n)is a substituted or unsubstituted alkyl or heteroalkyl chain, optionallywherein any carbon of the chain is substituted with an alkoxy, hydroxyl,alkylcarbonyloxy, alkyl-S—, thiol, alkyl-C(O)S—, amine, alkylamine,amide, or alkylamide; n is an integer selected from among the range of 2to 20; X is NH, O, S, absent, or a bond; L is independently absent, abond or a continuation of a bond, or a carbon comprising framework of 5to 200 atoms substituted at one or more atoms; r is an integer selectedfrom among 1, 2, 3 or 4; q is an integer selected from among 1, 2, 3 or4; z is an integer selected from among 1, 2, 3 or 4; and V isindependently absent, a bond or a continuation of a bond, anon-cleavable moiety or a conditionally-cleavable moiety; Y isindependently absent, a bond or a continuation of a bond, or a spacersystem which is comprised of 1 or more spacers; and Z is a moiety thatimproves pharmacokinetic properties, a therapeutic moiety or adiagnostic moiety, wherein Z is an organic compound that is electricallynegatively charged, hydrophobic and/or that has a molecular weight of atleast 400 g/mol; and wherein Formula IVb is:(Q)-NH—(C)_(n)—X-L-(V—(Y-(M)_(z))_(q))_(r)  Formula IVb or apharmaceutically acceptable salt or solvate thereof, wherein: Q isglutamine residue present in an antibody or antibody fragment; (C)_(n)is a substituted or unsubstituted alkyl or heteroalkyl chain, optionallywherein any carbon of the chain is substituted with a alkoxy, hydroxyl,alkylcarbonyloxy, alkyl-S—, thiol, alkyl-C(O)S—, amine, alkylamine,amide, or alkylamide; n is an integer selected from among the range of 2to 20; X is NH, O, S, absent, or a bond; L is independently absent, abond or a continuation of a bond, or a carbon comprising framework of 1to 200 atoms substituted at one or more atoms, optionally, wherein thecarbon comprising framework comprises a linear framework of 3 to 30carbon atoms optionally substituted at one or more atoms, optionallywherein the carbon comprising framework is a linear hydrocarbon, asymmetrically or asymmetrically branched hydrocarbon, monosaccharide,disaccharide, linear or branched oligosaccharide (asymmetricallybranched or symmetrically branched), other natural linear or branchedoligomers (asymmetrically branched or symmetrically branched), or adimer, trimer, or higher oligomer (linear, asymmetrically branched orsymmetrically branched) resulting from any chain-growth or step-growthpolymerization process; r is an integer selected from among 1, 2, 3 or4; q is an integer selected from among 1, 2, 3 or 4; z is an integerselected from among 1, 2, 3 or 4; V is independently absent, a bond or acontinuation of a bond, a non-cleavable moiety or aconditionally-cleavable moiety; Y is independently absent, a bond or acontinuation of a bond, or a spacer system which is comprised of 1 ormore spacers; M is independently: R or(RR′)-L′-(V′—(Y′—(Z)_(z′))_(q′))_(r′), wherein R is a reactive moiety;(RR′) is an addition product between R and a complementary reactivemoiety R′; L′ is independently absent, a bond or a continuation of abond, or a carbon comprising framework of 1 to 200 atoms substituted atone or more atoms, optionally, wherein the carbon comprising frameworkcomprises a linear framework of 3 to 30 carbon atoms optionallysubstituted at one or more atoms, optionally wherein the carboncomprising framework is a linear hydrocarbon, a symmetrically orasymmetrically branched hydrocarbon, monosaccharide, disaccharide,linear or branched oligosaccharide (asymmetrically branched orsymmetrically branched), other natural linear or branched oligomers(asymmetrically branched or symmetrically branched), or a dimer, trimer,or higher oligomer (linear, asymmetrically branched or symmetricallybranched) resulting from any chain-growth or step-growth polymerizationprocess; V′ is independently absent, a bond or a continuation of a bond,a non-cleavable moiety or a conditionally-cleavable moiety; Y′ isindependently absent, a bond or a continuation of a bond, or a spacersystem which is comprised of 1 or more spacers; Z is independently areactive group, a moiety that improves the pharmacokinetic properties, atherapeutic or diagnostic moiety, and each Z is directly coupled toeither Y or V when Y is absent, or L when both Y and V are absent; andz′, q′ and r′ are each independently an integer selected from among 1,2, 3 or
 4. 44. The method of claim 43, wherein n and m are equal andwherein m and n are 1, 2 or
 4. 45. A kit comprising a compound of claim41 and a TGase.
 46. A pharmaceutical composition comprising an antibodyof claim 1, and a pharmaceutically acceptable carrier.
 47. Apharmaceutical composition comprising an antibody of claim 25, and apharmaceutically acceptable carrier.
 48. A method of treating a diseasecomprising administering to a mammal a composition of claim
 46. 49. Amethod of treating a disease comprising administering to a mammal acomposition of claim
 47. 50. The antibody of claim 1, wherein Z is acytotoxic anti-cancer agent.
 51. The antibody of claim 50, wherein Z thecytotoxic anti-cancer agent is selected from the group consisting oftaxanes, anthracyclines, camptothecins, epothilones, mytomycins,combretastatins, vinca alkaloids, nitrogen mustards, maytansinoids,calicheamycins, duocarmycins, tubulysines, amatoxins, dolastatins andauristatins, enediynes, pyrrolobenzodiazepines, and ethylenimines.
 52. Amethod for preparing an antibody or antibody fragment comprising amoiety of interest (Z) bound thereto, comprising the steps of: (a)immobilizing an antibody or antibody fragment of Formula IVb on a solidsupport to provide an immobilized antibody, optionally comprising a stepof applying an antibody-containing sample to a solid support; (b)reacting the immobilized antibody or antibody fragment of step (a) witha compound of Formula III to generate an antibody-moiety-of-interestconjugate; wherein Formula IVb is:(Q)-NH—(C)_(n)—X-L-(V—(Y-(M)_(z))_(q))_(r)  Formula IVb or apharmaceutically acceptable salt or solvate thereof, wherein: Q isglutamine residue present in an antibody or antibody fragment; (C)_(n)is a substituted or unsubstituted alkyl or heteroalkyl chain, optionallywherein any carbon of the chain is substituted with a alkoxy, hydroxyl,alkylcarbonyloxy, alkyl-S—, thiol, alkyl-C(O)S—, amine, alkylamine,amide, or alkylamide; n is an integer selected from among the range of 2to 20; X is NH, O, S, absent, or a bond; L is independently absent, abond or a continuation of a bond, or a carbon comprising framework of 1to 200 atoms substituted at one or more atoms, optionally, wherein thecarbon comprising framework comprises a linear framework of 3 to 30carbon atoms optionally substituted at one or more atoms, optionallywherein the carbon comprising framework is a linear hydrocarbon, asymmetrically or asymmetrically branched hydrocarbon, monosaccharide,disaccharide, linear or branched oligosaccharide (asymmetricallybranched or symmetrically branched), other natural linear or branchedoligomers (asymmetrically branched or symmetrically branched), or adimer, trimer, or higher oligomer (linear, asymmetrically branched orsymmetrically branched) resulting from any chain-growth or step-growthpolymerization process; r is an integer selected from among 1, 2, 3 or4; q is an integer selected from among 1, 2, 3 or 4; z is an integerselected from among 1, 2, 3 or 4; V is independently absent, a bond or acontinuation of a bond, a non-cleavable moiety or aconditionally-cleavable moiety; Y is independently absent, a bond or acontinuation of a bond, or a spacer system which is comprised of 1 ormore spacers; M is independently: R or(RR′)-L′-(V′—(Y′—(Z)_(z′))_(q′))_(r′), wherein R is a reactive moiety;(RR′) is an addition product between R and a complementary reactivemoiety R′; L′ is independently absent, a bond or a continuation of abond, or a carbon comprising framework of 1 to 200 atoms substituted atone or more atoms, optionally, wherein the carbon comprising frameworkcomprises a linear framework of 3 to 30 carbon atoms optionallysubstituted at one or more atoms, optionally wherein the carboncomprising framework is a linear hydrocarbon, a symmetrically orasymmetrically branched hydrocarbon, monosaccharide, disaccharide,linear or branched oligosaccharide (asymmetrically branched orsymmetrically branched), other natural linear or branched oligomers(asymmetrically branched or symmetrically branched), or a dimer, trimer,or higher oligomer (linear, asymmetrically branched or symmetricallybranched) resulting from any chain-growth or step-growth polymerizationprocess; V′ is independently absent, a bond or a continuation of a bond,a non-cleavable moiety or a conditionally-cleavable moiety; Y′ isindependently absent, a bond or a continuation of a bond, or a spacersystem which is comprised of 1 or more spacers; Z is independently areactive group, a moiety that improves the pharmacokinetic properties, atherapeutic or diagnostic moiety, and each Z is directly coupled toeither Y or V when Y is absent, or L when both Y and V are absent; andz′, q′ and r′ are each independently an integer selected from among 1,2, 3 or 4; and wherein Formula III is:R′-L-(V—(Y-(M)_(z))_(q))_(r)  Formula III or a pharmaceuticallyacceptable salt or solvate thereof, wherein: R′ is a reactive group; Lis independently absent, or a carbon comprising framework of 1 to 200atoms substituted at one or more atoms, optionally, wherein the carboncomprising framework comprises a linear framework of 5 to 30 atomsoptionally substituted at one or more atoms, optionally wherein thecarbon comprising framework is a linear hydrocarbon, a symmetrically orasymmetrically branched hydrocarbon, monosaccharide, disaccharide,linear or branched oligosaccharide (asymmetrically branched orsymmetrically branched), other natural linear or branched oligomers(asymmetrically branched or symmetrically branched), or a dimer, trimer,or higher oligomer (linear, asymmetrically branched or symmetricallybranched) resulting from any chain-growth or step-growth polymerizationprocess; V is independently absent, a bond or a continuation of a bond,a non-cleavable moiety or a conditionally-cleavable moiety; Y isindependently absent, a bond or a continuation of a bond, or a spacersystem which is comprised of 1 or more spacers; M is independently: R or(RR′)-L′-(V′—(Y′—(Z)_(z′))_(q′))_(r′), wherein R is a reactive moiety;(RR′) is an addition product between an R and a complementary reactivegroup R′; L′ is independently absent, a bond or a continuation of abond, or a carbon comprising framework of 1 to 200 atoms substituted atone or more atoms, optionally, wherein the carbon comprising frameworkcomprises a linear framework of 3 to 30 carbon atoms optionallysubstituted at one or more atoms, optionally wherein the carboncomprising framework is a linear hydrocarbon, a symmetrically orasymmetrically branched hydrocarbon, monosaccharide, disaccharide,linear or branched oligosaccharide (asymmetrically branched orsymmetrically branched), other natural linear or branched oligomers(asymmetrically branched or symmetrically branched), or a dimer, trimer,or higher oligomer (linear, asymmetrically branched or symmetricallybranched) resulting from any chain-growth or step-growth polymerizationprocess; V′ is independently absent, a bond or a continuation of a bond,a non-cleavable moiety or a conditionally-cleavable moiety; Y′ isindependently absent, a bond or a continuation of a bond, or a spacersystem which is comprised of 1 or more spacers; Z is independently areactive group, a moiety that improves the pharmacokinetic properties, atherapeutic or diagnostic moiety, and each Z is directly coupled toeither Y or V when Y is absent, or L when both Y and V are absent; andz′, q′ and r′ are each independently an integer selected from among 1,2, 3 or
 4. 53. The method of claim 52, further comprising a step ofrecovering unreacted compound of Formula III and re-applying saidcompound of Formula III to the solid support to provide for highercompletion of the reaction between antibody comprising reactive group(R) and compound comprising reactive group (R′).
 54. An engineeredFc-containing polypeptide conjugate comprising the formula:(Fc-containing polypeptide)-T-A, wherein T is an acyl donorglutamine-containing tag engineered at a specific site or comprises anendogenous glutamine (Q) made reactive by the Fc-containing polypeptideengineering; wherein A is an amine donor agent; and wherein the aminedonor agent is site-specifically conjugated to the acyl donorglutamine-containing tag or the endogenous glutamine at a carboxylterminus, an amino terminus, or at an another site in the Fc-containingpolypeptide.
 55. The engineered Fc-containing polypeptide conjugate ofclaim 54, wherein: T comprises an endogenous glutamine made reactive bythe Fc-containing polypeptide engineering; and the amine donor agent issite-specifically conjugated to the endogenous glutamine at a carboxylterminus, an amino terminus, or at an another site in the Fc-containingpolypeptide.
 56. The engineered Fc-containing polypeptide conjugate ofclaim 54, wherein: (Fc-containing polypeptide)-T comprises an Fc regionof an antibody or antibody fragment containing a glutamine residue; andA is a linking reagent comprising an amine.
 57. The engineeredFc-containing polypeptide conjugate of claim 54, wherein the acyl donorglutamine-containing tag comprises an amino acid sequence XXQX, whereinX is any amino acid.
 58. The engineered Fc-containing polypeptideconjugate of claim 54, wherein the Fc-containing polypeptide conjugatecomprises a full length antibody heavy chain and an antibody lightchain.
 59. The engineered Fc-containing polypeptide conjugate of claim58, wherein the acyl donor glutamine-containing tag is located at thecarboxyl terminus of a heavy chain.
 60. The engineered Fc-containingpolypeptide conjugate of claim 58, wherein the acyl donorglutamine-containing tag is located at the carboxyl terminus of a lightchain.
 61. The engineered Fc-containing polypeptide conjugate of claim54, wherein the Fc-containing polypeptide comprises an antibody, whereinthe antibody is a monoclonal antibody, a polyclonal antibody, a humanantibody, a humanized antibody, a chimeric antibody, a bispecificantibody, a minibody, or an antibody fragment.
 62. The engineeredFc-containing polypeptide conjugate of claim 61, wherein the antibody isan IgG.
 63. The engineered Fc-containing polypeptide conjugate of claim54, wherein the amine donor agent comprises the formula:X—Y—Z, wherein X is an amine donor unit; Y is a linker; and Z is anagent moiety.
 64. The engineered Fc-containing polypeptide conjugate ofclaim 63, wherein the agent moiety is a cytotoxic agent.
 65. Theengineered Fc-containing polypeptide conjugate of claim 64, wherein thecytotoxic agent is selected from the group consisting of ananthracycline, an auristatin, a dolastatin, a duocarmycin, an enediyne,a maytansine, a taxane, a vinca alkaloid, and a tubulysin.
 66. Anengineered Fab-containing polypeptide conjugate comprising the formula:(Fab-containing polypeptide)-T-A, wherein T is an acyl donorglutamine-containing tag engineered at a specific site or comprises anendogenous glutamine made reactive by the Fab-containing polypeptideengineering; wherein A is an amine donor agent; wherein the amine donoragent is a biocompatible polymer comprising a reactive amine; andwherein the biocompatible polymer is site-specifically conjugated to theacyl donor glutamine-containing tag or the endogenous glutamine at acarboxyl terminus, an amino terminus, or at an another site in theFab-containing polypeptide.
 67. The engineered Fab-containingpolypeptide conjugate of claim 66, wherein: T comprises an endogenousglutamine made reactive by the Fab-containing polypeptide engineering;and wherein the biocompatible polymer is site-specifically conjugated tothe endogenous glutamine at a carboxyl terminus, an amino terminus, orat an another site in the Fab-containing polypeptide.
 68. The engineeredFab-containing polypeptide conjugate of claim 66, wherein:(Fab-containing polypeptide)-T comprises a Fab region of an antibody orantibody fragment containing a glutamine residue; and A is a linkingreagent comprising an amine.
 69. An engineered toxin polypeptideconjugate comprising the formula: (toxin polypeptide)-T-A, wherein T isan acyl donor glutamine-containing tag engineered at a specific site orcomprises an endogenous glutamine made reactive by the toxin polypeptideengineering; wherein A is an amine donor agent; wherein the amine donoragent is a biocompatible polymer comprising a reactive amine; andwherein the biocompatible polymer is site-specifically conjugated to theacyl donor glutamine-containing tag or the endogenous glutamine at acarboxyl terminus, an amino terminus, or at an another site in the toxinpolypeptide.
 70. The engineered toxin polypeptide conjugate of claim 69,wherein: T comprises an endogenous glutamine made reactive by the toxinpolypeptide engineering; and wherein the biocompatible polymer issite-specifically conjugated to the endogenous glutamine at a carboxylterminus, an amino terminus, or at an another site in the toxinpolypeptide.
 71. The engineered toxin polypeptide conjugate of claim 69,wherein: (toxin polypeptide)-T comprises an antibody or antibodyfragment containing a glutamine residue; and A is a linking reagentcomprising an amine.
 72. An engineered toxin polypeptide conjugatecomprising the formula: (toxin polypeptide)-T-B, wherein T is an acyldonor glutamine-containing tag engineered at a specific site; wherein Bis a biocompatible polymer; wherein the toxin polypeptide issite-specifically conjugated to the acyl donor glutamine-containing tagat any site in the biocompatible polymer.
 73. The engineered toxinpolypeptide conjugate of claim 72, wherein: (toxin polypeptide)-Tcomprises an antibody or antibody fragment containing a glutamineresidue; and B is a linking reagent comprising a biocompatible polymer.74. A composition comprising the engineered Fc-containing polypeptideconjugate of claim
 54. 75. A composition comprising the engineeredFab-containing polypeptide conjugate of claim
 66. 76. A compositioncomprising the engineered toxin polypeptide conjugate of claim
 69. 77. Apharmaceutical composition comprising the engineered Fc-containingpolypeptide conjugate of claim 54, and a pharmaceutically acceptableexcipient.
 78. A pharmaceutical composition comprising the engineeredFab-containing polypeptide conjugate of claim 66, and a pharmaceuticallyacceptable excipient.
 79. A pharmaceutical composition comprising theengineered toxin polypeptide conjugate of claim 69, and apharmaceutically acceptable excipient.
 80. A method for preparing theengineered Fc-containing polypeptide conjugate of claim 54, comprisingthe steps of: a) providing an engineered (Fc-containing polypeptide)-Tmolecule comprising the Fc-containing polypeptide located at the acyldonor glutamine-containing tag or the endogenous glutamine; b)contacting the amine donor agent with the engineered (Fc-containingpolypeptide)-T molecule in the presence of a transglutaminase; and c)allowing the engineered (Fc-containing polypeptide)-T to covalently linkto the amine donor agent to form the engineered Fc-containingpolypeptide conjugate.
 81. The method of claim 80, comprising the stepsof: a) providing an engineered (Fc-containing polypeptide)-T moleculecomprising the Fc-containing polypeptide located at the endogenousglutamine; b) contacting the amine donor agent with the engineered(Fc-containing polypeptide)-T molecule in the presence of atransglutaminase; and c) allowing the engineered (Fc-containingpolypeptide)-T to covalently link to the amine donor agent to form theengineered Fc-containing polypeptide conjugate.
 82. The method of claim80, wherein: (Fc-containing polypeptide)-T comprises an Fc region of anantibody or antibody fragment containing a glutamine residue.
 83. Themethod of claim 80, wherein the engineered Fc-containing polypeptideconjugate has conjugation efficiency of at least about 51%.
 84. Themethod of claim 80, wherein the amine donor agent has the formula:X—Y—Z, wherein X is an amine donor unit, Y is a linker, and Z is anagent moiety.
 85. A method for preparing an engineered Fab-containingpolypeptide conjugate of claim 66, comprising the steps of: a) providingan engineered (Fab-containing polypeptide)-T molecule comprising theFab-containing polypeptide located at the acyl donorglutamine-containing tag or the endogenous glutamine; b) contacting thebiocompatible polymer with the engineered (Fab-containing polypeptide)-Tmolecule in the presence of a transglutaminase; and c) allowing theengineered (Fab-containing polypeptide)-T to covalently link to thebiocompatible polymer to form the engineered Fab-containing polypeptideconjugate.
 86. The method of claim 85, comprising the steps of a)providing an engineered (Fab-containing polypeptide)-T moleculecomprising the Fab-containing polypeptide located at the endogenousglutamine; b) contacting the biocompatible polymer with the engineered(Fab-containing polypeptide)-T molecule in the presence of atransglutaminase; and c) allowing the engineered (Fab-containingpolypeptide)-T to covalently link to the biocompatible polymer to formthe engineered Fab-containing polypeptide conjugate.
 87. The method ofclaim 85, wherein (Fab-containing polypeptide)-T comprises a Fab regionof an antibody or antibody fragment containing a glutamine residue. 88.A method for preparing an engineered toxin polypeptide conjugate ofclaim 69, comprising the steps of: a) providing an engineered (toxinpolypeptide)-T molecule comprising the toxin polypeptide located at theacyl donor glutamine-containing tag or the endogenous glutamine; b)contacting the biocompatible polymer with the engineered (toxinpolypeptide)-T molecule in the presence of a transglutaminase; and c)allowing the engineered (toxin polypeptide)-T to covalently link to thebiocompatible polymer to form the engineered toxin polypeptideconjugate.
 89. The method of claim 88, comprising the steps of a)providing an engineered (toxin polypeptide)-T molecule comprising thetoxin polypeptide located at the endogenous glutamine; b) contacting thebiocompatible polymer with the engineered (toxin polypeptide)-T moleculein the presence of a transglutaminase; and c) allowing the engineered(toxin polypeptide)-T to covalently link to the biocompatible polymer toform the engineered toxin polypeptide conjugate.
 90. The method of claim88, wherein (toxin polypeptide)-T comprises an antibody or antibodyfragment containing a glutamine residue.